Compositions and methods for reducing neointima formation

ABSTRACT

Compositions, devices, grafts and methods for reducing or preventing anti-neointima following cardiovascular injuries and interventions are disclosed. The compositions, devices, and grafts typically include an effective amount of a CTP synthase 1 inhibitor to reduce proliferation of vascular smooth muscle cells, without substantial reducing the proliferation of endothelial cells. Methods of reducing neointima formation, accelerating re-endothelialization, and reducing restenosis in a subject using the compositions, devices, and grafts are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International ApplicationNo. PCT/US2014/037955 filed May 14, 2014, which claims benefit of U.S.Provisional Application No. 61/823,163, filed May 14, 2013, and thecontents of each of which are incorporated by reference herein in itsentireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under Agreement HL107526awarded by the National Institutes of Health. The Government has certainrights in the invention.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted as a text file named“UGA_2021_PCT_ST25.txt,” created on May 13, 2014, and having a size of14,231 bytes is hereby incorporated by reference pursuant to 37 C.F.R. §1.52(e)(5).

FIELD OF THE INVENTION

The field of the invention is generally related to compositions,devices, and methods for enhancing recovery from vascular injury orsurgery, and reducing morbidity and mortality associated with neointimaand restenosis.

BACKGROUND OF THE INVENTION

Neointimal hyperplasia is one of the major obstacles limiting thelong-term clinical efficiency of cardiovascular intervention includingangioplasty, bypass, and transplantation arteriopathy, etc.,(Degertekin, et al., Circulation., 106:1610-1613 (2002)). Neointimaformation also contributes to the development and progression of severalproliferative cardiovascular diseases such as atherosclerosis,hypertension, and diabetic vascular complications (Frank, et al., Curr.Opin. Lipidol., 15:523 (2004)). Under pathological conditions, vascularinjury causes denudation of endothelial layer, which triggers a seriesof acute and chronic inflammatory responses characterized by theproduction of various different growth factors or inflammatory cytokines(Murakami, et al., Am J Physiol Lung Cell Mol Physiol., 272:L197-L202(1997); Cotran, et al., J Am Soc Nephrol., 1:225-235 (1990)). Medialayer smooth muscle cell (SMC) proliferation and migration in responseto the injury-induced factors (such as platelet-derived growth factor,or PDGF) are essential events contributing to subsequent neointimalthickening (Fingerle, et al., Proc Natl Acad Sci., 86:8412 (1989);Clowes, et a., Circ. Res., 56:139-145 (1985)) which eventually leads tovessel narrowing. Re-endothelialization halts neointima formation andinitiates the successful vascular repair (Bauters, et al., Prog.Cardiovasc. Dis., 40:107-116 (1997); Kinlay, et al., Curr. Opin.Lipidol., 12:383 (2001)). However, currently available anti-neointimaldrugs indiscriminately block the proliferation of both SMCs andendothelial cells (EC), leading to impaired re-endothelialization andprolonged wound healing process. There remains a need to develop ananti-proliferation strategy that is SMC-sensitive.

Therefore, it is an object of the invention to provide compositions,devices, grafts, and methods of use thereof for reducing or preventingsmooth muscle cell proliferation in a subject.

It is a further object of the invention to provide compositions,devices, grafts, and methods of use thereof for promoting or enhancingre-endothelialization in a subject.

It is also an object of the invention to provide compositions, methods,and devices for reducing or preventing neointima formation, restenosis,or a combination thereof in a subject.

SUMMARY OF THE INVENTION

Compositions, devices, grafts, and methods for reducing or preventinganti-neointima following cardiovascular injuries and interventions aredisclosed. The compositions, devices, grafts, and methods are effectiveto reduce proliferation of vascular smooth muscle cells, withoutsubstantially reducing the proliferation of endothelial cells.Accordingly, re-endothelialization is accelerated in treated subjects.

The compositions typically include one or more cytidine-5′-triphosphatesynthase 1 (also referred to as CTP synthase 1 and CTPS1) inhibitors inan amount effective to reduce proliferation of vascular smooth musclecells (VSMC) in a subject. Preferably, the CTPS1 inhibitor reduces VSMCproliferation to a greater degree than the inhibitor reduces endothelialcell proliferation in the subject. In some embodiments, the compositiondoes not substantially reduce the proliferation of endothelial cells inthe subject. The compositions can include an effective amount of CTPS1inhibitor to reduce neointima formation, permit or promotere-endothelialization, or a combination thereof at a site of vascularinjury, a site of surgery, or a site of implantation of a vascularimplant in the subject.

Disclosed CTPS1 inhibitors include nucleoside analogs such ascyclopentenyl cytosine, 3-deazauridine (3-DU), carbodine; glutamineanalogs such as 6-diazo-5-oxo-L-norleucin (DON), and acivicin;functional nucleic acids designed to reduce expression of the CTPS1 geneor a gene product thereof; and polypeptides that reduces expression ofthe CTPS1 gene or a gene product thereof. In some embodiments the CTPS1inhibitor is an antisense molecule, siRNA, miRNA, aptamers, ribozymes,triplex forming molecules, RNAi, or external guide sequences that targetSEQ ID NO:1, or a gene editing composition such as CRISPR/Cas, zincfinger nuclease, or TALEN compositions that target the CTPS1 gene andreduce or otherwise modify its expression.

In some embodiments, the composition includes a delivery vehicle fordelivering the CTPS1 inhibitor to vascular smooth muscle cells. Thedelivery vehicle can be, for example, nanoparticles, microparticles,micelles, synthetic lipoprotein particles, liposomes, or carbonnanotubes.

The composition can include a targeting signal for enhancing delivery ofthe CTPS1 inhibitor to vascular smooth muscle cells. The targetingsignal can facilitate binding of the composition to smooth muscle cellsby targeting a cell surface ligand such as Tissue Factor or α_(v)β₃integrin; or the targeting signal can target the compositions to thevicinity of vascular injuries by binding to a marker of clots orthrombosis such as fibrin, gpIIb/IIIa, tissue factor/VIIA complex,activated clotting factor Xa, activated clotting factor IXa, or thefibrin condensation product d-dimer. The targeting signal can beoperably linked to the CTPS1 inhibitor or to the delivery vehicle.

Medical devices that are coated with or otherwise incorporate acomposition including a CTPS1 inhibitor are also disclosed. Discloseddevices include, but are not limited to, implants, needles, cannulas,catheters, shunts, stents, balloons, and valves. In preferredembodiments, the device is a stent, for example, a drug eluting stentthat elutes a composition including a CTPS1 inhibitor. The CTPS1inhibitor can increase re-endothelialization at the site of interventionand reduce or prevent stenosis or restenosis.

Vascular grafts that are coated with or otherwise incorporate acomposition including a CTPS1 inhibitor are also disclosed. The vasculargraft can be autologous, preserved autologous, allogeneic, xenogenic orsynthetic. Ex vivo treatment of the graft with a CTPS1 inhibitor priorto implantation can increase re-endothelialization at the site ofsurgery and reduce or prevent stenosis or restenosis.

Methods of reducing proliferation of vascular smooth muscle cells,methods of reducing or preventing neointima formation, and methods forpromoting re-endothelialization in a subject are also disclosed. Themethods typically include administering to the subject a composition,device, or graft that includes an effective amount of CTPS1 inhibitor toreduce proliferation of smooth muscle cells. The subject can haverestenosis or another vascular proliferation disorder, or have beenidentified as being at risk for restenosis or another vascularproliferation disorder. In some embodiments, the subject has undergone,is undergoing, or will undergo vascular trauma, angioplasty, vascularsurgery, or transplantation arteriopathy.

Some methods include co-administering to the subject one or moreadditional therapeutic agents. The additional therapeutic agents can beadditional anti-neointima agents, chemotherapeutic agents, antibodies,antibiotics, antivirals, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents,immunosuppressants, cytokines, chemokines, and growth factors.

In some embodiments, the methods include co-administering to the subjectcytidine or a cytidine analog.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a bar graph showing CTPS1 mRNA expression (fold change) incontrol, and PDGF-BB treated (5 ng/ml, 10 ng/ml, 20 ng/ml or 40 ng/ml)proliferating smooth muscle cells (SMC). FIG. 1B is a bar graph showingquantification of CTPS1 and PCNA protein expression in control, andPDGF-BB treated (5 ng/ml, 10 ng/ml) proliferating smooth muscle cells(SMC) (shown normalized to α-tubulin). FIGS. 1C and 1D are bar graphsshowing time dependent protein expression of CTPS1 (1C) and PCNA (1D) inrat carotid arteries over time (days) following injury (shown normalizedto α-tubulin). *P<0.05, **P<0.01, ***P<0.001 (n=3). FIG. 1E is a bargraph showing the relative mRNA expression levels of CTPS2 in Rat AorticSmooth Muscle Cells (RASMCs) treated with increasing concentrations ofPDGF-BB.

FIG. 2A is a bar graph showing the relative proliferation rate (%) ofcontrol SMC and SMC treated with 1 nM, 20 nM, or 500 nM of the CTPSinhibitor CPEC. FIG. 2B is a bar graph showing PCNA protein level (foldchange) in control SMC and SMC treated with 500 nM CPEC for 24 hours(normalized to α-tubulin). FIG. 2C is a bar graph showing the relativeproliferation rate (%) of control SMC and SMC infected with or withoutPDGF-BB, adenoviral expressed GFP (Ad-GFP), and/or adenoviral expressedshRNA against CTPS1 (Ad-shCTPS1). FIG. 2D is a bar graph showing CTPS1and PCNA expression (fold change) in control SMC and SMC transfectedwith adenoviral expressed GFP (Ad-GFP) or adenoviral expressed shRNAagainst CTPS1 (Ad-shCTPS1) (normalized to α-tubulin). *P<0.05, **P<0.01,***P<0.001 (n=4).

FIG. 3A is a bar graph showing the relative migration distance ofcontrol SMC and SMC treated with 20 nM CPEC or 500 nM CPEC. FIG. 3B is agraph showing the relative migration distance of control SMC and SMCtreated with 10 ng/ml PDGF-BB and infected with adenoviral expressed GFP(Ad-GFP) or adenoviral expressed shRNA against CTPS1 (Ad-shCTPS1).*P<0.05, **P<0.01, ***P<0.001 (n=4).

FIG. 4A is a series of scatter plots showing the results flow cytometryanalysis of SMC apoptosis (propidium iodide vs. Annexin V) in NC:negative control (top-left panel); PC: positive control (top-rightpanel); vehicle-treated cells (Control) (bottom-left panel); and cellstreated with 500 nM CPEC (bottom-right panel). FIG. 4B is a series ofplots showing the results of flow cytometry analysis of SMCproliferation (cell number vs. DNA content) for control and 500 nM CPECtreat cells. FIG. 4C is a series of plots showing the results of flowcytometry analysis of SMC proliferation (cell number vs. DNA content)for cells infected with adenoviral expressed GFP (Ad-GFP) or adenoviralexpressed shRNA against CTPS1 (Ad-shCTPS1). FIG. 4D is a bar graphshowing relative CDK1 activation (phosphorylated CDK1/total CDK1) incontrol SMC and SMC serum-starved, or treated with 20 nM or 500 nM CPEC.FIG. 4E is a bar graph showing relative CDK1 activation (phosphorylatedCDK1/total CDK1) in cells infected with adenoviral expressed GFP(Ad-GFP) or adenoviral expressed shRNA against CTPS1 (Ad-shCTPS1) withor without PDGF-BB treatment. *P<0.05, **P<0.01, ***P<0.001 (n=4).

FIG. 5A is a series of micrographs showing neointima formation (elastin(VG) staining) in control and blocked balloon injured arteries of micetreated with saline, 1 mg/kg CPEC, or 2 mg/kg CPEC. Images show arteries14 days after injury. FIG. 5B is a bar graph showing relative neointimaformation in blocked balloon injured arteries of mice treated withsaline, 1 mg/kg CPEC, or 2 mg/kg CPEC (quantification of FIG. 5A). FIG.5C a bar graph showing relative neointima formation in blocked ballooninjured arteries of mice infected with adenoviral expressed GFP (Ad-GFP)or adenoviral expressed shRNA against CTPS1 (Ad-shCTPS1). *P<0.05,**P<0.01, ***P<0.001 (n=5).

FIG. 6A is a bar graph showing the relative proliferation rate (%) ofsmooth muscle cells (SMC) and endothelial cells (EC) untreated (control)or treated with 5 nM CPEC, 20 nM CPEC, 500 nM CPEC, or 500 nM CPEC+10 μMcytidine. FIG. 6B is a bar graph showing quantification ofre-endothelialization (%) in wire-injured mouse arteries for uninjuredanimals (control) and saline or CPEC treated animals 3 and 5 days afterinjury. *P<0.05, **P<0.01, ***P<0.001 (n=5). FIG. 6C is a bar graphshowing the relative mRNA expression levels of CTPS2 in proliferationendothelial cells (C166 cells) when treated with various concentrationsof PDGF-BB, VEGF, or 10% FBS. FIG. 6D is a bar graph showing therelative proliferation rate (%) of endothelial cells (EC) and smoothmuscle cells (SMC) in the presence or absence of abundant (10 μM)cytidine. FIG. 6E is a bar graph showing the relative proliferation rate(%) of endothelial cells (EC) and smooth muscle cells (SMC) withincreasing concentrations of paclitaxel (0 nM, 20 nM, 500 nM) and 500 nMpaclitaxel with 10 μM cytidine. FIG. 6F is a bar graph showingre-endothelialization (%) relative to control (day 14) of injured ratstreated with saline or 1 mg/kg CPEC, *P<0.05, **P<0.01, ***P<0.001(n=5).

FIG. 7A is a bar graph showing mRNA expression (relative expressionlevel) of salvage pathway-related genes (UCK1, UCK2, CMPK, NME1, andNME2) in ECs (C166) and SMCs (VSMC) with or without the addition of CPECand cytidine. FIG. 7B is a bar graph showing quantification of NME1 andNME2 protein expression was induced in ECs by CPEC in the presence ofboth cytidine and growth factors (PDGF-BB or VEGF) (normalized toα-tubulin). FIG. 7C is a bar graph showing relative proliferation ratein EC infected with adenoviral expressed GFP (Ad-GFP) or adenoviralexpressed shRNA against CTPS1 (Ad-shCTPS1), with or withoutCPEC+cytidine. *P<0.05, **P<0.01, ***P<0.001 (n=5). FIG. 7D is a bargraph showing the relative expression of NME1 mRNA in control (Ad-GFP)and NME1 overexpressing (Ad-NME1) cells as assayed by qPCR, *P<0.05,**P<0.01, ***P<0.001 (n=3). FIG. 7E is a graph showing relativeexpression of NME2 mRNA in control (Ad-GFP), NME2 knockdown (Ad-shNME2)and NME2 overexpressing (Ad-NME2) cells as assayed by qPCR, *P<0.05,**P<0.01, ***P<0.001 (n=3). FIG. 7F is a bar graph showing the relativecytidine utilization rate of control cells (Ad-GFP) and cellsoverexpressing NME1 (Ad-NME1), NME2 (Ad-NME2), or NME1+NME2(Ad-NME1+Ad-NME2), and treated with CPEC, and with or without cytidine,*P<0.05, **P<0.01, **P<0.001 (n=8). FIG. 7G is a bar graph showing therelative density of NME2 staining in control and injured arteriestreatment undergoing vascular repair treated with or without CPEC. FIG.7H is a bar graph showing the relative percentage of CD31-positive cellsin injured arteries, *P<0.05, **P<0.01, ***P<0.001 (n=5).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences, as determined by comparing the sequences. In theart, “identity” also means the degree of sequence relatedness betweenpolypeptide as determined by the match between strings of suchsequences. “Identity” and “similarity” can be readily calculated byknown methods, including, but not limited to, those described in(Computational Molecular Biology, Lesk, A. M., Ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., Eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991;and Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988).Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs. Thepercent identity between two sequences can be determined by usinganalysis software (i.e., Sequence Analysis Software Package of theGenetics Computer Group, Madison Wis.) that incorporates the Needelmanand Wunsch, (J. Mol. Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST,and XBLAST). The default parameters are used to determine the identityfor the polypeptides of the present disclosure.

By way of example, a polypeptide sequence may be identical to thereference sequence, that is be 100% identical, or it may include up to acertain integer number of amino acid alterations as compared to thereference sequence such that the % identity is less than 100%. Suchalterations are selected from: at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofamino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in the reference polypeptideby the numerical percent of the respective percent identity (divided by100) and then subtracting that product from said total number of aminoacids in the reference polypeptide.

“Pharmaceutically acceptable carrier” encompasses any of the standardpharmaceutical carriers, such as a phosphate buffered saline solution,water and emulsions such as an oil/water or water/oil emulsion, andvarious types of wetting agents.

“Inhibit” or other forms of the word such as “inhibiting” or“inhibition” means to hinder or restrain a particular characteristic. Itis understood that this is typically in relation to some standard orexpected value, in other words it is relative, but that it is not alwaysnecessary for the standard or relative value to be referred to. Forexample, “inhibits CTP synthase 1” means hindering or restraining theactivity of the enzyme relative to a standard or a control. “InhibitsCTP synthase 1” can also mean to hinder or restrain the synthesis orexpression of the enzyme relative to a standard or control.

“Treatment” or “treating” means to administer a composition to a subjector a system with an undesired condition (e.g., restenosis or othervascular proliferative disorder). The condition can include a disease.“Prevention” or “preventing” means to administer a composition to asubject or a system at risk for the condition. The condition can includea predisposition to a disease. The effect of the administration of thecomposition to the subject (either treating and/or preventing) can be,but is not limited to, the cessation of a particular symptom of acondition, a reduction or prevention of the symptoms of a condition, areduction in the severity of the condition, the complete ablation of thecondition, a stabilization or delay of the development or progression ofa particular event or characteristic, or minimization of the chancesthat a particular event or characteristic will occur. It is understoodthat where treat or prevent are used, unless specifically indicatedotherwise, the use of the other word is also expressly disclosed.

“Subject,” “individual,” and “patient” refer to any individual who isthe target of treatment using the disclosed compositions. The subjectcan be a vertebrate, for example, a mammal Thus, the subject can be ahuman. The subjects can be symptomatic or asymptomatic. The term doesnot denote a particular age or sex. Thus, adult and newborn subjects,whether male or female, are intended to be covered. A subject caninclude a control subject or a test subject.

“Operably linked” refers to a juxtaposition wherein the components areconfigured so as to perform their usual function. For example, controlsequences or promoters operably linked to a coding sequence are capableof effecting the expression of the coding sequence, and an organellelocalization sequence operably linked to protein will direct the linkedprotein to be localized at the specific organelle.

“Localization Signal or Sequence or Domain or Ligand” or “TargetingSignal or Sequence or Domain or Ligand” are used interchangeably andrefer to a signal that directs a molecule to a specific cell, tissue,organelle, or intracellular region. The signal can be polynucleotide,polypeptide, or carbohydrate moiety or can be an organic or inorganiccompound sufficient to direct an attached molecule to a desiredlocation.

A “graft” is a tissue for transplantation. This may be in the form ofcells or non-dissociated tissue. It may or may not have been treatedprior to implantation to sterilize, modify, or cleanse the graft. Graftsinclude autograft, allograft, and synthetic tissues and organs, tissuesproduced by tissue engineering and non-biological medical devices byattachment of specific ligands (i.e. counter ligands attached to eachsurface) or by electrostatic or other non-covalent means.

“Microparticles” refers to particles having a diameter between onemicron and 1000 microns, typically less than 400 microns, more typicallyless than 100 microns, most preferably for the uses described herein inthe range of less than 10 microns in diameter. Microparticles includemicrocapsules and microspheres unless otherwise specified.

“Nanoparticles” refer to particles having a diameter of less than onemicron, more typically between 50 and 1000 nanometers, preferably in therange of 100 to 300 nanometers.

“Implantation” refers to placement of a graft within the body. This maybe by surgical means or minimally invasive means such as a catheter orby injection or infusion into a tissue.

II. Compositions for Reducing or Preventing Neointima Formation

Drug-eluting stents (DES) are a common treatment for coronary arterydiseases. However, anti-proliferative drugs currently used in the clinicsuch as sirolimus-(Cypher) and paclitaxel-(Taxus) have side effectscausing defective re-endothelialization and increasing risk of latethrombosis. It has been discovered that blockade of CTPS1 functionreduces smooth muscle cell (SMC) proliferation and acceleratesre-endothelialization.

Proliferative SMCs are known to produce various paracrine factors suchas endostatin and thrombospondin to inhibit endothelial cellproliferation. Therefore, reduction of proliferating SMCs also benefitsre-endothelialization as long as endothelial cell proliferation is notalso inhibited. It has been discovered that this can be achieved byactivating the CTP salvage pathway. Salvage pathway enzymes such as NME2appear to be expressed at a relatively low level in normal endothelialcell growth conditions. However, when CTPS activity is blocked, NME2 isdramatically induced in endothelial cells while neointima proliferatingSMCs are significantly reduced. The combined effect of blocking CTPS1 oninhibiting SMC proliferation while sustaining endothelial cellproliferation results in the accelerated re-endothelialization inendothelial cell-denuded vessels.

Compositions for preventing or reducing neointima formation andpromoting re-endothelialization by blocking CTPS1 activity duringvascular remodeling are disclosed.

A. CTPS Inhibitors

The compositions disclosed herein typically include an inhibitor ofcytidine-5′-triphosphate synthase 1 (CTPS1) that blocks, reduces, orinhibits expression, activity, or bioavailability of CTPS1. CTP synthase1 (also referred to as CTP synthetase and CTPS1) is a metabolic enzymethat catalyzes CTP biosynthesis from UTP, ATP and glutamine, anessential step for DNA and RNA synthesis during cell proliferation(Ostrander, J. Biol. Chem., 273:18992-19001 (1998); Long, J. Biol.Chem., 245:80-87 (1970)). The enzyme is important in the biosynthesis ofphospholipids and nucleic acids, and plays a role in cell growth,development, and tumorigenesis.

It has been discovered that CTPS1 is induced smooth muscle cells (SMCs),but not endothelial cells (ECs) during vascular remodeling. Therefore,reducing CTPS1 function can suppresses SMC proliferation and neointimaformation while promoting re-endothelialization in injured vessels.CTPS1 function can be reduced by blocking, reducing, or inhibiting CTPS1gene expression or expression of a CTPS1 gene product. CTPS1 can also bereduced by blocking, reducing, or inhibiting activity of CTPS1 enzymaticactivity. CTPS1 can also be reduced by reducing or inhibiting thebioavailability or bioactivity of CTPS1 for example by increasingturnover or degradation of CTPS1 mRNA or protein.

A number of compounds that block, reduce, or inhibit CTPS1 expression oractivity, and dosages and formulations thereof are known in the art anddiscussed in more detail below. Some of the compounds have exhibitedsome toxicity to subjects in pre-clinical and clinical trials. It isbelieved, however, that the dosage for reducing neointima formation andpromoting re-endothelialization can be lower than the dosages fortreating cancer. As discussed in more detail below, in some embodimentsthe compositions are delivered locally to the site of treatment, whichreduces toxicity associated with systemic delivery.

Classes of CTP synthase inhibitors discussed below include nucleosideanalogues, glutamine analogs, and functional nucleic acids.

1. Nucleoside Analog Inhibitors of CTPS1

The CTPS1 inhibitor can be a nucleoside or nucleotide analog.

a. Cyclopentenyl Cytosine

In some embodiments the nucleoside or nucleotide analog is cyclopentenylcytosine (CPEC), or a derivative, analogue or prodrug, or apharmacologically active salt thereof. CPEC is an analogue of cytidinein which the ribose moiety is substituted by a carbocyclic sugar. CPEChas the structure:

Cyclopentenyl cytosine (CPEC, or CPE-C) is converted to the activemetabolite cyclopentenyl cytosine 5′-triphosphate (CPEC-TP); CPEC-TPcompetitively inhibits cytidine triphosphate (CTP) synthase, therebydepleting intracellular cytidine pools and inhibiting DNA and RNAsynthesis. CPEC has been proposed as an anticancer and antiviraltherapy. See, for example, Schimmel, et al., Cyclopentenyl Cytosine(Cpec): An Overview of its in vitro and in vivo activity,” Chapter 2 inCurrent Cancer Drug Targets 7:325-334 (2007), which is specificallyincorporated herein by reference in its entirety.

Other cyclopentenyl cytosine based analogs are also known in the art.See, for example, U.S. Published Application No. 2009/0270431, which isspecifically incorporated by reference in its entirety and provides ahydroxyl protected cyclopentenyl cytosine analog, aprotected-2′-fluoro-cyclopentenyl cytosine analog, and others.

CPEC has been the subject of clinical trials for the treatment ofcancer. Formulations and dosages for treating human subjects are knownin the art. See, for example, Politi, et al., Cancer Chemother.Pharmacol., 36(6):513-23 (1995) and Schimmel, et al., Drug Dev. Ind.Pharm., 32(4):497-503 (2006), Tanaka and Sata, Arterioscler Thromb VascBiol., 33(10):2286-7 (2013) all of which are specifically incorporatedby reference in their entireties.

b. 3-Deazauridine (3-DU)

In some embodiments the nucleoside or nucleotide analog is3-deazauridine (3-DU), or a derivative, analog or prodrug, or apharmacologically active salt thereof 3-DU is a uridine analog havingthe structure:

3-DU is not incorporated into nucleic acids. However, after itsconversion to 3-deazauridine 5′-triphosphate, it produces a potentinhibition of CTP synthetase (McPartland, et al., Cancer Res., 34:3107-3111 (1974)). Inhibition of CTPS1 by the nucleotide form of 3-DUproduces a marked depletion of the intracellular pool of CTP and dCTP(Brockman, Ann. NY. Acad. Sci., 225:501-521 (1975), and has been testedas an anti-cancer and anti-viral therapy (Momparler, et al., CancerRes., 39(10):3822-7 (1979), Gao, et al., Nucleosides, NucleotidesNucleic Acids, 19:371-377 (2000)).

Formulations and dosages including 3-DU are discussed in U.S. Pat. No.3,836,645, and analogs thereof are discussed in Lalut, et al., Bioorg.Med. Chem. Lett., 22(24):7461-4 (2012), WO/2011/070152, McNamara, etal., JBC, 33(7):2006-11 (1990), and Legraverend, et al., Nucleosides andNucleotides, 5(2):125-134 (1986), each of which is specificallyincorporated by reference herein in its entirety.

c. Carbodine

In some embodiments the nucleoside or nucleotide analog is carbodine, ora derivative, analog or prodrug, or a pharmacologically active saltthereof. Carbodine is a nucleoside analog of cytidine. The structuresfor (−)-carbodine, (+)-carbodine are:

Carbodine is termed a “carbocyclic analog” which refers to nucleosideanalogs in which the tetrahydrofuran ring of the parent nucleoside isreplaced by the cyclopentane ring (Shealy, et al., J. Am. Chem. Soc.,88:3885-3887 (1966)). Carbodine, related carbocyclic analogs, andformulations and dosages thereof are known in the art and have beentested for their antiviral activity. See, for example, Shannon, et al.,Antimicrobial Agents and Chemotherapy, 20(6):769-776 (1981),Georges-Courbot, et al., Agents Chemother., 50:1768-1772 (2006),Julander, et al., Antiviral Res., 80(3):309-15 (2008), and U.S. Pat. No.8,242,120, each of which is specifically incorporated by referenceherein in its entirety.

2. Glutamine Analog Inhibitors CTPS1

The CTPS1 inhibitor can be a glutamine analog or antagonist. Largeneutral amino acid L-glutamine antagonists include6-diazo-5-oxo-L-norleucine (L-DON), andL-[αS,5S]-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, alsoknown as acivicin, are described in Ahluwalia, G. S., et al, Pharmac.Ther., 42(16):243-271 (1990), which is specifically incorporated byreference herein in its entirety.

a. 6-diazo-5-oxo-L-norleucin (DON)

In some embodiments the glutamine analog or antagonist is6-diazo-5-oxo-L-norleucin (DON), or a derivative, analog or prodrug, ora pharmacologically active salt thereof. DON is a glutamine antagonist,which was isolated originally from Streptomyces in a sample of Peruviansoil. DON has the structure:

Due to its similarity to glutamine, DON can enter catalytic centers ofglutamine utilizing enzymes such as CTP synthase, and inhibits them byalkylation. DON has been the subject of limited clinical trials since1957 for the treatment of cancer and more recently infectious diseases(see, for example, Catane, et al., Cancer Treat. Rep., 63, 1033-1038(1979); and Ahluwalia, et al. Pharmacol. Ther. 46, 243-271 (1990)). U.S.Pat. No. 2,965,634 describes norleucine derivatives, such as DON, and aprocess for the production thereof. Dosages and formulations are alsoknown in the art. See, for example, Sullivan, et al., Cancer ChemotherPharmacol., 21(1):78-84 (1988), Hofer, et al., PNAS, 98(11):6412-6416(2001), Carcamo, et al., PLoS ONE, 6(12): e29690.doi:10.1371/journal.pone.0029690 (2011), each of which is specificallyincorporated by reference herein in its entirety. For example, forL-DON, the dosage can range from 0.1-1.1 mg/kg/day for oraladministration and from 0.2-0.6 mg/kg/day for intramuscular orintravenous administration.

b. Acivicin

In some embodiments the glutamine analog or antagonist isL-[αS,5S]-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid(acivicin), or a derivative, analog or prodrug, or a pharmacologicallyactive salt thereof. Acivicin has the structure:

3-bromoacivicin has a profile similar to that of acivicin in testsagainst a panel of tumor cell lines (Conti, et al., Farmaco,58(9):683-90 (2003), and was found to be a CTP synthetase inhibitorthree times as potent as its 3-chloro analog (Conti, et al, Chem MedChem, 6(2): 329-333 (2011)), each of which are specifically incorporatedby reference herein in its entirety.

Acivicin has been the subject of clinical trials for the treatment ofcancer. Dosages and formulations are known in the art, see, for example,Hidalgo, Clinical Cancer Research, 4(11): 2763-2770 (1998), U.S. Pat.Nos. 3,856,807, 3,878,047, and 5,087,639 each of which is specificallyincorporated by reference herein in its entirety. For example, foracivicin, the dosage can range from 12-25 mg/square meter of bodysurface per day and administration is carried out intravenously as abolus injection or by continuous infusion. Acivicin is also described inU.S. Pat. No. 5,489,562.

3. Other Compounds that Inhibit CTPS1

Other suitable compounds are described in WO 2014/170435, and include,for example, gemcitabine (2′,2′-difluorodeoxycytidine, dFdC),actinomycin D, cycloheximide, dibutyryl cyclic AMP, and 6-azauridine.

4. Functional Nucleic Acids Inhibitors of CTPS1

The CTPS1 inhibitor can be a functional nucleic acid. Functional nucleicacids are nucleic acid molecules that have a specific function, such asbinding a target molecule or catalyzing a specific reaction. Asdiscussed in more detail below, functional nucleic acid molecules can bedivided into the following non-limiting categories: antisense molecules,siRNA, miRNA, aptamers, ribozymes, triplex forming molecules, RNAi, andexternal guide sequences. The functional nucleic acid molecules can actas effectors, inhibitors, modulators, and stimulators of a specificactivity possessed by a target molecule, or the functional nucleic acidmolecules can possess a de novo activity independent of any othermolecules.

Functional nucleic acid molecules can interact with any macromolecule,such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functionalnucleic acids can interact with the mRNA or the genomic DNA of a targetpolypeptide or they can interact with the polypeptide itself. Oftenfunctional nucleic acids are designed to interact with other nucleicacids based on sequence homology between the target molecule and thefunctional nucleic acid molecule. In other situations, the specificrecognition between the functional nucleic acid molecule and the targetmolecule is not based on sequence homology between the functionalnucleic acid molecule and the target molecule, but rather is based onthe formation of tertiary structure that allows specific recognition totake place.

Therefore the compositions can include one or more functional nucleicacids designed to reduce expression of the CTPS1 gene, or a gene productthereof.

a. CTPS1 Sequences

In some embodiments, the composition includes a functional nucleic acidor polypeptide designed to target and reduce or inhibit expression ortranslation of CPTS1 mRNA; or to reduce or inhibit expression, reduceactivity, or increase degradation of CPTS1 protein. In some embodiments,the composition includes a vector suitable for in vivo expression of thefunctional nucleic acid.

Nucleic acid and amino acid sequences for CTPS1 are known in the art.See, for example, NCBI Reference Sequence: NM_001905.2, Homo sapiens CTPsynthase 1 (CTPS1), mRNA, which provides the nucleic acid sequence:

(SEQ ID NO: 1)   1 tccctcgccc ggaggcagag atgcgctggc gcatcaccgc caggagccca cagtgaaaga  61 ccatcggatg gaaggcgacg cccagaactc caggagaccg ctgtgggagg acgcgaggcc  121 aggtgacgaa taggccaggc gtgagttccc aaacagcctc ctccccttca agagagtaag  181 cttgggccac aggctgggac ggaagcagag gggcagacac gccaccaccc gcccggcctc  241 gaacctacgg cggcacagtt cagcggaggc ggcccagcgg tcctgtcccg cgcctgcgca  301 ctccaggccc cgccccgccc cgcgccctcc aggcccggcc cgccctccaa cctctgcgtg  361 cgcacagcct agagcccgcc tccgtgaaag actgccgggc gcatgcggtc ggggttgttc  421 actggctgtc cggggctccg cgcgcgtcgc cggcccagct ctgtcgctga cgggaggatc  481 tgaagccggc cgcaggtcaa agagtaaaat gaagtacatt ctggttactg gtggtgttat  541 atcaggaatt ggaaaaggaa tcattgccag cagtgtgggc acaatactca agtcatgtgg  601 tttacatgta acttcaatca aaattgaccc ctacattaac attgatgcag gaacattctc  661 tccttatgag catggtgagg tttttgtgct ggatgatggt ggggaagtag accttgacct  721 gggtaactat gagcggttcc ttgacatccg cctcaccaag gacaataatc tgaccactgg  781 aaagatatac cagtatgtca ttaacaagga acggaaagga gattacttgg ggaaaactgt  841 ccaagttgtc cctcatatca cagatgcaat ccaggagtgg gtgatgagac aggcgttaat  901 acctgtagat gaagatggcc tggaacctca agtgtgt tt attgagcttg gtggaaccgt  961 gggggacata gaaagcatgc cctttattga ggccttccgt cagttccaat tcaaggtcaa 1021 aagagagaac ttttgtaaca tccacgtcag tctagttccc cagccaagtt caacagggga 1081 acagaagact aaacctaccc agaatagtgt tcgggaactt agaggacttg ggctttcccc 1141 agatctggtt gtatgcaggt gctcaaatcc acttgacaca tcagtgaagg agaaaatatc 1201 aatgttctgc catgttgagc ctgaacaagt gatctgtgtc cacgatgtct catccatcta 1261 ccgagtcccc ttgttgttag aggagcaagg ggttgtagat tattttcttc gaagacttga 1321 ccttcctatt gagaggcagc caagaaaaat gctgatgaaa tggaaagaga tggctgacag 1381 atatgatcgc ttgctggaga cctgctctat tgcccttgtg ggcaaataca cgaagttctc 1441 agactcctat gcctctgtca ttaaggctct ggagcattct gcactggcca tcaaccacaa 1501 attggaaatc aagtacatag attctgcgga cttggagccc atcacctcgc aagaagagcc 1561 cgtgcgctac cacgaagctt ggcagaagct ctgtagtgct catggagtgc tggttccagg 1621 aggatttggt gttcgaggaa cagaaggaaa aatccaagca attgcctggg ctcggaatca 1681 gaaaaagcct tttttgggcg tgtgcttagg gatgcagttg gcagtggttg aattctcaag 1741 aaacgtgctg ggatggcaag atgccaattc tacagagttt gaccctacga ccagtcatcc 1801 cgtggtcgta gacatgccag aacacaaccc agggcagatg ggcggaacca tgaggctggg 1861 caagaggaga accctgttcc agaccaagaa ctcagtcatg aggaaactct atggagacgc 1921 agactacttg gaagagaggc accgccaccg atttgaggtg aatccagtct ggaaaaagtg 1981 tttggaagaa caaggcttga agtttgttgg ccaagatgtt gaaggagaga gaatggaaat 2041 tgtggagtta gaagatcatc ccttttttgt tggggttcag taccaccctg agttcctgtc 2101 caggcctatc aagccctccc caccatactt tggcctcctc ctggcctctg tggggcggct 2161 ctcacattac ctccagaaag gctgcaggct ctcacccagg gacacctata gtgacaggag 2221 tggaagcagc tcccctgact ctgaaatcac cgaactgaag tttccatcaa taaatcatga 2281 ctgatcttgt agcggatgat tcttcaagag acccttcaaa cttgggtaga gtttacagct 2341 ctgactttac actcggcttt ggagactttc tttaaattat gtttttatta agattatttt 2401 attatgcgga aaggtatttg ggaaacttgt cacttgcatg tcccatcacg tgtactggct 2461 cctctgtggt gtctgcctgt tgcgtgacac tctccttgca gttcttgagt tgcggcagaa 2521 catcgcgatg ggaaccgatg gtgggtgggg ctgcagagtg ccccatcggt caccttgttt 2581 ctcaactacc tcgcatcatt gcagatgcta gcgcgttgcc tgtcgctttc ccttggatac 2641 ctagaccgtt ataaagtgtg ccacatggac ttaccgagca tggagagagg attttagcta 2701 ggatttgaac acttggtgct gggaacctca gggtattgct tgccactaag ccatgaaacc 2761 agagacaaaa tctctatact gccctgagtt ggggggaatt ctcagtgcca actgtggctg 2821 gtcctcattc aaagggacgg tcagtttggt gtcaacatga aacaccaaga tgtctgtctc 2881 tgaagcgtga ttttaaaatc cccatgcctg tggctgcgct tcctatttct agggctggga 2941 aacactcctt gcatcaaggg gtcacttaca gaacaaagaa tcttttgggg gaaacttcct 3001 ctaaaaccct ctcatatata gacagctttg actggagggt ccatttttct tccaggatgg 3061 tgttactgca gttgaaaggg caatatgaag ttactttctt aatgtgacct agcaataggc 3121 atagctacgt ggcactatat tctggccaga ctcgatgtgt actctaactt aagaaataaa 3181 tcagtaaggc agaacaagaa aaaaaaaaaa aaaaaaa, and the amino acid sequence

(SEQ ID NO: 2) MKYILVTGGV ISGIGKGIIA SSVGTILKSC GLHVISIKIDPYINIDAGTF SPYEHGEVFV LDDGGEVDLD LGNYERFLDIRLIKDNNLTT GKIYQYVINK ERKGDYLGKT VQVVPHITDAIQEWVMRQAL IPVDEDGLEP QVCVIELGGT VGDIESMPFIEAFRQFQFKV KRENFCNIHV SLVPQPSSTG EQKTKPTQNSVRELRGLGLS PDLVVCRCSN PLDTSVKEKI SMFCHVEPEQVICVHDVSSI YRVPLLLEEQ GVVDYFLRRL DLPIERQPRKMLMKWKEMAD RYDRLLETCS IALVGKYTKF SDSYASVIKALEHSALAINH KLEIKYIDSA DLEPITSQEE PVRYHEAWQKLCSAHGVLVP GGFGVRGTEG KIQAIAWARN QKKPFLGVCLGMQLAVVEFS RNVLGWQDAN STEFDPTTSH PVVVDMPEHNPGQMGGTMRL GKRRTLFQTK NSVMRKLYGD ADYLEERHRHRFEVNPVWKK CLEEQGLKFV GQDVEGERME IVELEDHPFFVGVQYHPEFL SRPIKPSPPY FGLLLASVGR LSHYLQKGCRLSPRDTYSDR SGSSSPDSEI TELKFPSINH D.

In some embodiments, a functional nucleic acid or polypeptide isdesigned to target a segment of the nucleic acid sequence of SEQ IDNO:1, or the complement thereof, or a genomic sequence correspondingtherewith, or variants thereof having a nucleic acid sequence at least65%, 70%_(,) 71%_(,) 72%_(,) 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 1.

In some embodiments, a functional nucleic acid or polypeptide isdesigned to target a segment of a the nucleic acid encoding the aminoacid sequence of SEQ ID NO:2, or the complement thereof, or variantsthereof having a nucleic acid sequence 65%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to anucleic acid encoding the amino acid sequence of SEQ ID NO:2.

In some embodiments, the function nucleic acid hybridizes to the nucleicacid of SEQ ID NO:1, or a complement thereof, for example, understringent conditions. In some embodiments, the functional nucleic acidhybridizes to a nucleic acid sequence that encodes SEQ ID NO:2, or acomplement thereof, for example, under stringent conditions.

b. Functional Nucleic Acids

i. Antisense

The functional nucleic acids can be antisense molecules. Antisensemolecules are designed to interact with a target nucleic acid moleculethrough either canonical or non-canonical base pairing. The interactionof the antisense molecule and the target molecule is designed to promotethe destruction of the target molecule through, for example, RNAse Hmediated RNA-DNA hybrid degradation. Alternatively the antisensemolecule is designed to interrupt a processing function that normallywould take place on the target molecule, such as transcription orreplication. Antisense molecules can be designed based on the sequenceof the target molecule. There are numerous methods for optimization ofantisense efficiency by finding the most accessible regions of thetarget molecule. Exemplary methods include in vitro selectionexperiments and DNA modification studies using DMS and DEPC. It ispreferred that antisense molecules bind the target molecule with adissociation constant (K_(d)) less than or equal to 10⁻⁶, 10⁻⁸, 10⁻¹⁰,or 10⁻¹².

ii. Aptamers

The functional nucleic acids can be aptamers. Aptamers are moleculesthat interact with a target molecule, preferably in a specific way.Typically aptamers are small nucleic acids ranging from 15-50 bases inlength that fold into defined secondary and tertiary structures, such asstem-loops or G-quartets. Aptamers can bind small molecules, such as ATPand theophiline, as well as large molecules, such as reversetranscriptase and thrombin. Aptamers can bind very tightly with K_(d)'sfrom the target molecule of less than 10⁻¹² M. It is preferred that theaptamers bind the target molecule with a K_(d) less than 10⁻⁶, 10⁻⁸,10⁻¹⁰, or 10⁻¹². Aptamers can bind the target molecule with a very highdegree of specificity. For example, aptamers have been isolated thathave greater than a 10,000 fold difference in binding affinities betweenthe target molecule and another molecule that differ at only a singleposition on the molecule. It is preferred that the aptamer have a K_(d)with the target molecule at least 10, 100, 1000, 10,000, or 100,000 foldlower than the K_(d) with a background binding molecule. It is preferredwhen doing the comparison for a molecule such as a polypeptide, that thebackground molecule be a different polypeptide.

iii. Ribozymes

The functional nucleic acids can be ribozymes. Ribozymes are nucleicacid molecules that are capable of catalyzing a chemical reaction,either intramolecularly or intermolecularly. It is preferred that theribozymes catalyze intermolecular reactions. There are a number ofdifferent types of ribozymes that catalyze nuclease or nucleic acidpolymerase type reactions which are based on ribozymes found in naturalsystems, such as hammerhead ribozymes. There are also a number ofribozymes that are not found in natural systems, but which have beenengineered to catalyze specific reactions de novo. Preferred ribozymescleave RNA or DNA substrates, and more preferably cleave RNA substrates.Ribozymes typically cleave nucleic acid substrates through recognitionand binding of the target substrate with subsequent cleavage. Thisrecognition is often based mostly on canonical or non-canonical basepair interactions. This property makes ribozymes particularly goodcandidates for target specific cleavage of nucleic acids becauserecognition of the target substrate is based on the target substratessequence.

iv. Triplex Forming Oligonucleotides

The functional nucleic acids can be triplex forming molecules. Triplexforming functional nucleic acid molecules are molecules that caninteract with either double-stranded or single-stranded nucleic acid.When triplex molecules interact with a target region, a structure calleda triplex is formed in which there are three strands of DNA forming acomplex dependent on both Watson-Crick and Hoogsteen base-pairing.Triplex molecules are preferred because they can bind target regionswith high affinity and specificity. It is preferred that the triplexforming molecules bind the target molecule with a K_(d) less than 10⁻⁶,10⁻⁸, 10⁻¹⁰, or 10⁻¹².

v. External Guide Sequences

The functional nucleic acids can be external guide sequences. Externalguide sequences (EGSs) are molecules that bind a target nucleic acidmolecule forming a complex, which is recognized by RNase P, which thencleaves the target molecule. EGSs can be designed to specifically targeta RNA molecule of choice. RNAse P aids in processing transfer RNA (tRNA)within a cell. Bacterial RNAse P can be recruited to cleave virtuallyany RNA sequence by using an EGS that causes the target RNA:EGS complexto mimic the natural tRNA substrate. Similarly, eukaryotic EGS/RNAseP-directed cleavage of RNA can be utilized to cleave desired targetswithin eukarotic cells. Representative examples of how to make and useEGS molecules to facilitate cleavage of a variety of different targetmolecules are known in the art.

vi. RNA Interference

In some embodiments, the functional nucleic acids induce gene silencingthrough RNA interference. Gene expression can also be effectivelysilenced in a highly specific manner through RNA interference (RNAi).This silencing was originally observed with the addition of doublestranded RNA (dsRNA) (Fire, et al. (1998) Nature, 391:806-11; Napoli, etal. (1990) Plant Cell 2:279-89; Hannon, (2002) Nature, 418:244-51). OncedsRNA enters a cell, it is cleaved by an RNase III-like enzyme, Dicer,into double stranded small interfering RNAs (siRNA) 21-23 nucleotides inlength that contains 2 nucleotide overhangs on the 3′ ends (Elbashir, etal. (2001) Genes Dev., 15:188-200; Bernstein, et al. (2001) Nature,409:363-6; Hammond, et al. (2000) Nature, 404:293-6). In an ATPdependent step, the siRNAs become integrated into a multi-subunitprotein complex, commonly known as the RNAi induced silencing complex(RISC), which guides the siRNAs to the target RNA sequence (Nykanen, etal. (2001) Cell, 107:309-21). At some point the siRNA duplex unwinds,and it appears that the antisense strand remains bound to RISC anddirects degradation of the complementary mRNA sequence by a combinationof endo and exonucleases (Martinez, et al. (2002) Cell, 110:563-74).However, the effect of iRNA or siRNA or their use is not limited to anytype of mechanism.

Short Interfering RNA (siRNA) is a double-stranded RNA that can inducesequence-specific post-transcriptional gene silencing, therebydecreasing or even inhibiting gene expression. In one example, a siRNAtriggers the specific degradation of homologous RNA molecules, such asmRNAs, within the region of sequence identity between both the siRNA andthe target RNA. For example, WO 02/44321 discloses siRNAs capable ofsequence-specific degradation of target mRNAs when base-paired with 3′overhanging ends, herein incorporated by reference for the method ofmaking these siRNAs.

Sequence specific gene silencing can be achieved in mammalian cellsusing synthetic, short double-stranded RNAs that mimic the siRNAsproduced by the enzyme dicer (Elbashir, et al. (2001) Nature, 411:494498) (Ui-Tei, et al. (2000) FEBS Lett 479:79-82). siRNA can bechemically or in vitro-synthesized or can be the result of shortdouble-stranded hairpin-like RNAs (shRNAs) that are processed intosiRNAs inside the cell. Synthetic siRNAs are generally designed usingalgorithms and a conventional DNA/RNA synthesizer. Suppliers includeAmbion (Austin, Tex.), ChemGenes (Ashland, Mass.), Dharmacon (Lafayette,Co.), Glen Research (Sterling, Va.), MWB Biotech (Esbersberg, Germany),Proligo (Boulder, Co.), and Qiagen (Vento, The Netherlands). siRNA canalso be synthesized in vitro using kits such as Ambion's SILENCER® siRNAConstruction Kit.

The production of siRNA from a vector is more commonly done through thetranscription of a short hairpin RNAse (shRNAs). Kits for the productionof vectors comprising shRNA are available, such as, for example,Imgenex's GENESUPPRESSOR™ Construction Kits and Invitrogen's BLOCK-IT™inducible RNAi plasmid and lentivirus vectors.

In some embodiment, the functional nucleic acid is siRNA, shRNA, miRNA.In some embodiments, the composition includes a vector expressing thefunctional nucleic acid. Methods of making and using vectors for in vivoexpression of functional nucleic acids such as antisenseoligonucleotides, siRNA, shRNA, miRNA, EGSs, ribozymes, and aptamers areknown in the art.

vii. Other Gene Editing Compositions

In some embodiments the functional nucleic acids are gene editingcompositions. Gene editing compositions can include nucleic acids thatencode an element or elements that induce a single or a double strandbreak in the target cell's genome, and optionally a polynucleotide. Thecompositions can be used, for example, to reduce or otherwise modifyexpression of CTPS1.

1. Strand Break Inducing Elements

CRISPR/Cas

In some embodiments, the element that induces a single or a doublestrand break in the target cell's genome is a CRISPR/Cas system. CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats) is anacronym for DNA loci that contain multiple, short, direct repetitions ofbase sequences. The prokaryotic CRISPR/Cas system has been adapted foruse as gene editing (silencing, enhancing or changing specific genes)for use in eukaryotes (see, for example, Cong, Science,15:339(6121):819-823 (2013) and Jinek, et al., Science, 337(6096):816-21(2012)). By transfecting a cell with the required elements including aCas gene and specifically designed CRISPRs, the organism's genome can becut and modified at any desired location. Methods of preparingcompositions for use in genome editing using the CRISPR/Cas systems aredescribed in detail in WO 2013/176772 and WO 2014/018423, which arespecifically incorporated by reference herein in their entireties.

In general, “CRISPR system” refers collectively to transcripts and otherelements involved in the expression of or directing the activity ofCRISPR-associated (“Cas”) genes, including sequences encoding a Casgene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or anactive partial tracrRNA), a tracr-mate sequence (encompassing a “directrepeat” and a tracrRNA-processed partial direct repeat in the context ofan endogenous CRISPR system), a guide sequence (also referred to as a“spacer” in the context of an endogenous CRISPR system), or othersequences and transcripts from a CRISPR locus. One or more tracr matesequences operably linked to a guide sequence (e.g., directrepeat-spacer-direct repeat) can also be referred to as pre-crRNA(pre-CRISPR RNA) before processing or crRNA after processing by anuclease.

In some embodiments, a tracrRNA and crRNA are linked and form a chimericcrRNA-tracrRNA hybrid where a mature crRNA is fused to a partialtracrRNA via a synthetic stem loop to mimic the natural crRNA:tracrRNAduplex as described in Cong, Science, 15:339(6121):819-823 (2013) andJinek, et al., Science, 337(6096):816-21 (2012)). A single fusedcrRNA-tracrRNA construct can also be referred to as a guide RNA or gRNA(or single-guide RNA (sgRNA)). Within an sgRNA, the crRNA portion can beidentified as the ‘target sequence’ and the tracrRNA is often referredto as the ‘scaffold’.

There are many resources available for helping practitioners determinesuitable target sites once a desired DNA target sequence is identified.For example, numerous public resources, including a bioinformaticallygenerated list of about 190,000 potential sgRNAs, targeting more than40% of human exons, are available to aid practitioners in selectingtarget sites and designing the associate sgRNA to affect a nick ordouble strand break at the site. See also, crispr.u-psud.fr/, a tooldesigned to help scientists find CRISPR targeting sites in a wide rangeof species and generate the appropriate crRNA sequences.

In some embodiments, one or more vectors driving expression of one ormore elements of a CRISPR system are introduced into a target cell suchthat expression of the elements of the CRISPR system direct formation ofa CRISPR complex at one or more target sites. While the specifics can bevaried in different engineered CRISPR systems, the overall methodologyis similar. A practitioner interested in using CRISPR technology totarget a DNA sequence (such as CTPS1) can insert a short DNA fragmentcontaining the target sequence into a guide RNA expression plasmid. ThesgRNA expression plasmid contains the target sequence (about 20nucleotides), a form of the tracrRNA sequence (the scaffold) as well asa suitable promoter and necessary elements for proper processing ineukaryotic cells. Such vectors are commercially available (see, forexample, Addgene). Many of the systems rely on custom, complementaryoligos that are annealed to form a double stranded DNA and then clonedinto the sgRNA expression plasmid. Co-expression of the sgRNA and theappropriate Cas enzyme from the same or separate plasmids in transfectedcells results in a single or double strand break (depending of theactivity of the Cas enzyme) at the desired target site.

Zinc Finger Nucleases

In some embodiments, the element that induces a single or a doublestrand break in the target cell's genome is a nucleic acid construct orconstructs encoding a zinc finger nucleases (ZFNs). ZFNs are typicallyfusion proteins that include a DNA-binding domain derived from azinc-finger protein linked to a cleavage domain.

The most common cleavage domain is the Type IIS enzyme Fokl. Foklcatalyzes double-stranded cleavage of DNA, at 9 nucleotides from itsrecognition site on one strand and 13 nucleotides from its recognitionsite on the other. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as well as Li et al. Proc., Natl. Acad. Sci. USA 89(1992):4275-4279; Li et al. Proc. Natl. Acad. Sci. USA, 90:2764-2768(1993); Kim et al. Proc. Natl. Acad. Sci. USA. 91:883-887 (1994a); Kimet al. J. Biol. Chem. 269:31, 978-31,982 (1994b). One or more of theseenzymes (or enzymatically functional fragments thereof) can be used as asource of cleavage domains.

The DNA-binding domain, which can, in principle, be designed to targetany genomic location of interest, can be a tandem array of Cys₂His₂ zincfingers, each of which generally recognizes three to four nucleotides inthe target DNA sequence. The Cys₂His₂ domain has a general structure:Phe (sometimes Tyr)-Cys-(2 to 4 amino acids)-Cys-(3 amino acids)-Phe(sometimes Tyr)-(5 amino acids)-Leu-(2 amino acids)-His-(3 aminoacids)-His. By linking together multiple fingers (the number varies:three to six fingers have been used per monomer in published studies),ZFN pairs can be designed to bind to genomic sequences 18-36 nucleotideslong.

Engineering methods include, but are not limited to, rational design andvarious types of empirical selection methods. Rational design includes,for example, using databases including triplet (or quadruplet)nucleotide sequences and individual zinc finger amino acid sequences, inwhich each triplet or quadruplet nucleotide sequence is associated withone or more amino acid sequences of zinc fingers which bind theparticular triplet or quadruplet sequence. See, for example, U.S. Pat.Nos. 6,140,081; 6,453,242; 6,534,261; 6,610,512; 6,746,838; 6,866,997;7,067,617; U.S. Published Application Nos. 2002/0165356; 2004/0197892;2007/0154989; 2007/0213269; and International Patent ApplicationPublication Nos. WO 98/53059 and WO 2003/016496.

Transcription Activator-Like Effector Nucleases

In some embodiments, the element that induces a single or a doublestrand break in the target cell's genome is a nucleic acid construct orconstructs encoding a transcription activator-like effector nuclease(TALEN). TALENs have an overall architecture similar to that of ZFNs,with the main difference that the DNA-binding domain comes from TALeffector proteins, transcription factors from plant pathogenic bacteria.The DNA-binding domain of a TALEN is a tandem array of amino acidrepeats, each about 34 residues long. The repeats are very similar toeach other; typically they differ principally at two positions (aminoacids 12 and 13, called the repeat variable diresidue, or RVD). Each RVDspecifies preferential binding to one of the four possible nucleotides,meaning that each TALEN repeat binds to a single base pair, though theNN RVD is known to bind adenines in addition to guanine. TAL effectorDNA binding is mechanistically less well understood than that ofzinc-finger proteins, but their seemingly simpler code could prove verybeneficial for engineered-nuclease design. TALENs also cleave as dimers,have relatively long target sequences (the shortest reported so farbinds 13 nucleotides per monomer) and appear to have less stringentrequirements than ZFNs for the length of the spacer between bindingsites. Monomeric and dimeric TALENs can include more than 10, more than14, more than 20, or more than 24 repeats.

Methods of engineering TAL to bind to specific nucleic acids aredescribed in Cermak, et al, Nucl. Acids Res. 1-11 (2011). U.S. PublishedApplication No. 2011/0145940, which discloses TAL effectors and methodsof using them to modify DNA. Miller et al. Nature Biotechnol 29: 143(2011) reported making TALENs for site-specific nuclease architecture bylinking TAL truncation variants to the catalytic domain of Foklnuclease. The resulting TALENs were shown to induce gene modification inimmortalized human cells. General design principles for TALE bindingdomains can be found in, for example, WO 2011/072246.

2. Gene Altering Polynucleotides

The nuclease activity of the genome editing systems described hereincleave target DNA to produce single or double strand breaks in thetarget DNA. Double strand breaks can be repaired by the cell in one oftwo ways: non-homologous end joining, and homology-directed repair. Innon-homologous end joining (NHEJ), the double-strand breaks are repairedby direct ligation of the break ends to one another. As such, no newnucleic acid material is inserted into the site, although some nucleicacid material may be lost, resulting in a deletion. In homology-directedrepair, a donor polynucleotide with homology to the cleaved target DNAsequence is used as a template for repair of the cleaved target DNAsequence, resulting in the transfer of genetic information from a donorpolynucleotide to the target DNA. As such, new nucleic acid material canbe inserted/copied into the site.

Therefore, in some embodiments, the genome editing compositionoptionally includes a donor polynucleotide. The modifications of thetarget DNA due to NHEJ and/or homology-directed repair can be used toinduce gene correction, gene replacement, gene tagging, transgeneinsertion, nucleotide deletion, gene disruption, gene mutation, etc.

Accordingly, cleavage of DNA by the genome editing composition can beused to delete nucleic acid material from a target DNA sequence bycleaving the target DNA sequence and allowing the cell to repair thesequence in the absence of an exogenously provided donor polynucleotide.Alternatively, if the genome editing composition includes a donorpolynucleotide sequence that includes at least a segment with homologyto the target DNA sequence, the methods can be used to add, i.e., insertor replace, nucleic acid material to a target DNA sequence (e.g., to“knock in” a nucleic acid that encodes for a protein, an siRNA, anmiRNA, etc.), to add a tag (e.g., 6×His, a fluorescent protein (e.g., agreen fluorescent protein; a yellow fluorescent protein, etc.),hemagglutinin (HA), FLAG, etc.), to add a regulatory sequence to a gene(e.g., promoter, polyadenylation signal, internal ribosome entrysequence (IRES), 2A peptide, start codon, stop codon, splice signal,localization signal, etc.), to modify a nucleic acid sequence (e.g.,introduce a mutation), and the like. As such, the compositions can beused to modify DNA in a site-specific, i.e., “targeted”, way, forexample gene knock-out, gene knock-in, gene editing, gene tagging, etc.as used in, for example, gene therapy.

In applications in which it is desirable to insert a polynucleotidesequence into a target DNA sequence, a polynucleotide including a donorsequence to be inserted is also provided to the cell. By a “donorsequence” or “donor polynucleotide” or “donor oligonucleotide” it ismeant a nucleic acid sequence to be inserted at the cleavage site. Thedonor polynucleotide typically contains sufficient homology to a genomicsequence at the cleavage site, e.g., 70%, 80%, 85%, 90%, 95%, or 100%homology with the nucleotide sequences flanking the cleavage site, e.g.,within about 50 bases or less of the cleavage site, e.g., within about30 bases, within about 15 bases, within about 10 bases, within about 5bases, or immediately flanking the cleavage site, to supporthomology-directed repair between it and the genomic sequence to which itbears homology. The donor sequence is typically not identical to thegenomic sequence that it replaces. Rather, the donor sequence maycontain at least one or more single base changes, insertions, deletions,inversions or rearrangements with respect to the genomic sequence, solong as sufficient homology is present to support homology-directedrepair. In some embodiments, the donor sequence includes anon-homologous sequence flanked by two regions of homology, such thathomology-directed repair between the target DNA region and the twoflanking sequences results in insertion of the non-homologous sequenceat the target region.

c. Oligonucleotide Composition

The functional nucleic acids can be DNA or RNA nucleotides whichtypically include a heterocyclic base (nucleic acid base), a sugarmoiety attached to the heterocyclic base, and a phosphate moiety whichesterifies a hydroxyl function of the sugar moiety. The principalnaturally-occurring nucleotides comprise uracil, thymine, cytosine,adenine and guanine as the heterocyclic bases, and ribose or deoxyribosesugar linked by phosphodiester bonds.

In some embodiments, the oligonucleotides are composed of nucleotideanalogs that have been chemically modified to improve stability,half-life, or specificity or affinity for a target receptor, relative toa DNA or RNA counterpart. The chemical modifications include chemicalmodification of nucleobases, sugar moieties, nucleotide linkages, orcombinations thereof. As used herein “modified nucleotide” or“chemically modified nucleotide” defines a nucleotide that has achemical modification of one or more of the heterocyclic base, sugarmoiety or phosphate moiety constituents. In some embodiments, the chargeof the modified nucleotide is reduced compared to DNA or RNAoligonucleotides of the same nucleobase sequence. For example, theoligonucleotide can have low negative charge, no charge, or positivecharge.

Typically, nucleoside analogs support bases capable of hydrogen bondingby Watson-Crick base pairing to standard polynucleotide bases, where theanalog backbone presents the bases in a manner to permit such hydrogenbonding in a sequence-specific fashion between the oligonucleotideanalog molecule and bases in a standard polynucleotide (e.g.,single-stranded RNA or single-stranded DNA). In some embodiments, theanalogs have a substantially uncharged, phosphorus containing backbone.

i. Heterocyclic Bases

The principal naturally-occurring nucleotides include uracil, thymine,cytosine, adenine and guanine as the heterocyclic bases. Theoligonucleotides can include chemical modifications to their nucleobaseconstituents. Chemical modifications of heterocyclic bases orheterocyclic base analogs may be effective to increase the bindingaffinity or stability in binding a target sequence. Chemically-modifiedheterocyclic bases include, but are not limited to, inosine,5-(1-propynyl) uracil (pU), 5-(1-propynyl) cytosine (pC),5-methylcytosine, 8-oxo-adenine, pseudocytosine, pseudoisocytosine, 5and 2-amino-5-(2′-deoxy-.beta.-D-ribofuranosyl)pyridine(2-aminopyridine), and various pyrrolo- and pyrazolopyrimidinederivatives.

ii. Sugar Modifications

Oligonucleotides can also contain nucleotides with modified sugarmoieties or sugar moiety analogs. Sugar moiety modifications include,but are not limited to, 2′-O-aminoetoxy, 2′-O-amonioethyl (2′-OAE),2′-O-methoxy, 2′-O-methyl, 2-guanidoethyl (2′-OGE), 2′-O,4′-C-methylene(LNA), 2′-O-(methoxyethyl) (2′-OME) and 2′-O—(N-(methyl)acetamido)(2′-OMA). 2′-O-aminoethyl sugar moiety substitutions are especiallypreferred because they are protonated at neutral pH and thus suppressthe charge repulsion between the TFO and the target duplex. Thismodification stabilizes the C3′-endo conformation of the ribose ordexyribose and also forms a bridge with the i−1 phosphate in the purinestrand of the duplex.

In some embodiments, the functional nucleic acid is a morpholinooligonucleotide. Morpholino oligonucleotides are typically composed oftwo more morpholino monomers containing purine or pyrimidinebase-pairing moieties effective to bind, by base-specific hydrogenbonding, to a base in a polynucleotide, which are linked together byphosphorus-containing linkages, one to three atoms long, joining themorpholino nitrogen of one monomer to the 5′ exocyclic carbon of anadjacent monomer. The purine or pyrimidine base-pairing moiety istypically adenine, cytosine, guanine, uracil or thymine. The synthesis,structures, and binding characteristics of morpholino oligomers aredetailed in U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506,5,166,315, 5,521,063, and 5,506,337.

Important properties of the morpholino-based subunits typically include:the ability to be linked in a oligomeric form by stable, unchargedbackbone linkages; the ability to support a nucleotide base (e.g.adenine, cytosine, guanine, thymidine, uracil or inosine) such that thepolymer formed can hybridize with a complementary-base target nucleicacid, including target RNA, with high T_(m), even with oligomers asshort as 10-14 bases; the ability of the oligomer to be activelytransported into mammalian cells; and the ability of an oligomer:RNAheteroduplex to resist RNAse degradation.

In some embodiments, oligonucleotides employ morpholino-based subunitsbearing base-pairing moieties, joined by uncharged linkages, asdescribed above.

iii. Internucleotide Linkages

Oligonucleotides connected by an internucleotide bond that refers to achemical linkage between two nucleoside moieties. Modifications to thephosphate backbone of DNA or RNA oligonucleotides may increase thebinding affinity or stability oligonucleotides, or reduce thesusceptibility of oligonucleotides nuclease digestion. Cationicmodifications, including, but not limited to, diethyl-ethylenediamide(DEED) or dimethyl-aminopropylamine (DMAP) may be especially useful dueto decrease electrostatic repulsion between the oligonucleotide and atarget. Modifications of the phosphate backbone may also include thesubstitution of a sulfur atom for one of the non-bridging oxygens in thephosphodiester linkage. This substitution creates a phosphorothioateinternucleoside linkage in place of the phosphodiester linkage.Oligonucleotides containing phosphorothioate internucleoside linkageshave been shown to be more stable in vivo.

Examples of modified nucleotides with reduced charge include modifiedinternucleotide linkages such as phosphate analogs having achiral anduncharged intersubunit linkages (e.g., Sterchak, E. P. et al., Organic.Chem., 52:4202, (1987)), and uncharged morpholino-based polymers havingachiral intersubunit linkages (see, e.g., U.S. Pat. No. 5,034,506), asdiscussed above. Some internucleotide linkage analogs includemorpholidate, acetal, and polyamide-linked heterocycles.

In another embodiment, the oligonucleotides are composed of lockednucleic acids. Locked nucleic acids (LNA) are modified RNA nucleotides(see, for example, Braasch, et al., Chem. Biol., 8(1):1-7 (2001)). LNAsform hybrids with DNA which are more stable than DNA/DNA hybrids, aproperty similar to that of peptide nucleic acid (PNA)/DNA hybrids.Therefore, LNA can be used just as PNA molecules would be. LNA bindingefficiency can be increased in some embodiments by adding positivecharges to it. Commercial nucleic acid synthesizers and standardphosphoramidite chemistry are used to make LNAs.

In some embodiments, the oligonucleotides are composed of peptidenucleic acids. Peptide nucleic acids (PNAs) are synthetic DNA mimics inwhich the phosphate backbone of the oligonucleotide is replaced in itsentirety by repeating N-(2-aminoethyl)-glycine units and phosphodiesterbonds are typically replaced by peptide bonds. The various heterocyclicbases are linked to the backbone by methylene carbonyl bonds. PNAsmaintain spacing of heterocyclic bases that is similar to conventionalDNA oligonucleotides, but are achiral and neutrally charged molecules.Peptide nucleic acids are comprised of peptide nucleic acid monomers.

Other backbone modifications include peptide and amino acid variationsand modifications. Thus, the backbone constituents of oligonucleotidessuch as PNA may be peptide linkages, or alternatively, they may benon-peptide peptide linkages. Examples include acetyl caps, aminospacers such as 8-amino-3,6-dioxaoctanoic acid (referred to herein asO-linkers), amino acids such as lysine are particularly useful ifpositive charges are desired in the PNA, and the like. Methods for thechemical assembly of PNAs are well known. See, for example, U.S. Pat.Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571and 5,786,571.

Oligonucleotides optionally include one or more terminal residues ormodifications at either or both termini to increase stability, and/oraffinity of the oligonucleotide for its target. Commonly used positivelycharged moieties include the amino acids lysine and arginine, althoughother positively charged moieties may also be useful. Oligonucleotidesmay further be modified to be end capped to prevent degradation using apropylamine group. Procedures for 3′ or 5′ capping oligonucleotides arewell known in the art.

In some embodiments, the functional nucleic acid can be single strandedor double stranded.

B. Targeting Signal or Domain

The compositions can be optionally modified to include one or moretargeting signals, ligands, or domains. The targeting signal can beoperably linked with the CTPS1 inhibitor, or a delivery vehicle such asa microparticle. For example, in some embodiments, the targeting signalis linked or conjugated directly or indirectly to the CTPS1 inhibitor.In some embodiments, the targeting signal is linked, conjugated, orassociated directly, or indirectly, with a delivery vehicle such as aliposome or a nanoparticle. Delivery vehicles are discussed in moredetail below. The targeting signal or sequence can be specific for ahost, tissue, organ, cell, organelle, non-nuclear organelle, or cellularcompartment.

In some embodiments, the targeting signal binds to its ligand orreceptor which is located on the surface of a target cell such as tobring the composition or a delivery vehicle thereof and cell membranessufficiently close to each other to allow penetration of the compositionor delivery vehicle into the cell. In a preferred embodiment, thetargeting molecule is selected from the group consisting of an antibodyor antigen binding fragment thereof, an antibody domain, an antigen, acell surface receptor, a cell surface adhesion molecule, a majorhistocompatibility locus protein, a viral envelope protein and a peptideselected by phage display that binds specifically to a defined cell.

Targeting the compositions or delivery vehicles to specific cells can beaccomplished by modifying the disclosed compositions or deliveryvehicles to express specific cell and tissue targeting signals. Thesesequences target specific cells and tissues, but in some embodiments theinteraction of the targeting signal with the cell does not occur througha traditional receptor: ligand interaction. Eukaryotic cells have anumber of distinct cell surface molecules. The structure and function ofeach molecule can be specific to the origin, expression, character andstructure of the cell. Determining the unique cell surface complement ofmolecules of a specific cell type can be determined using techniqueswell known in the art.

One skilled in the art will appreciate that the tropism of thecompositions or delivery vehicles described can be altered by merelychanging the targeting signal. In one specific embodiment, compositionsare provided that enable the addition of cell surface antigen specificantibodies to the composition or delivery vehicle for targeting thedelivery the CTPS1 inhibitor to the target cells.

It is known in the art that nearly every cell type in a tissue in amammalian organism possesses some unique cell surface receptor orantigen. Thus, it is possible to incorporate nearly any ligand for thecell surface receptor or antigen as a targeting signal. For example,peptidyl hormones can be used a targeting moieties to target delivery tothose cells which possess receptors for such hormones. Chemokines andcytokines can similarly be employed as targeting signals to targetdelivery of the complex to their target cells. A variety of technologieshave been developed to identify genes that are preferentially expressedin certain cells or cell states and one of skill in the art can employsuch technology to identify targeting signals which are preferentiallyor uniquely expressed on the target tissue of interest.

In preferred embodiments, the targeting signal or domain targets theCTPS1 inhibitor or a delivery vehicle carrying the inhibitor to thecells of the vasculature, preferably smooth muscle cells of thevasculature, more preferably media layer smooth muscle cells.

Compositions and methods for targeting the vasculature are known in theart. For example, compositions and delivery vehicles can be targeted toVSMC surface epitopes, for example, Tissue Factor (TF). Also referred toas also platelet tissue factor, factor III, thromboplastin, or CD142, TFis a protein present in subendothelial tissue and leukocytes necessaryfor the initiation of thrombin formation from the zymogen prothrombin.TF protein is typically described as having three domains: anextracellular domain, a transmembrane domain, and cytoplasmic domain. Ina preferred embodiment, the targeting signal binds to the extracellulardomain of TF. For example, the targeting signal can be an antibody orantigen binding fragment thereof that binds to the extracellular domainof TF.

The extracellular domain of TF binds factor VIIa. Factor VIIa is aprotein which consists of several domains. One of these domains, thecarboxylated GLA domain, binds in the presence of calcium to negativelycharged phospholipids. Binding of VIIa to negatively chargedphospholipids greatly enhances the protein-protein binding of VIIa toTF. In some embodiments, the targeting signal includes one or moredomains of Factors VIIa that bind to TF.

Compositions and methods for delivering agents to vascular smooth musclecells (VSMCs) by targeting Tissue Factor (TF) are known in the art. See,for example, Lanza, et al., Circulation, 106:2842-2847 (2002) and Lanza,et al., J. Am. Soc. Echo., 13:608-614 (2000), each of which isspecifically incorporated by references in its entirety, which describetargeting drugs and imaging reagents, respectively, to media VSMCs usingTF-targeting nanoparticles.

In another embodiment, the targeting signal binds to α_(v)β₃ integrin,which is expressed on the surface of stretch-activated smooth musclecells exposed along the sheared tissue planes of the arterial wallfollowing vascular damage (Cyrus, et al., Arteriosclerosis, Thrombosis,and Vascular Biology, 28:820-826 (2008)). Accordingly, the signalingagent can be an antibody that binds to α_(v)β₃-integrin, or a ligand ofα_(v)β₃-integrin, for example a protein or polypeptide including theamino acid sequence R-G-D, or a small molecule agonist or antagonist ofα_(v)β₃-integrin.

In some embodiments, the compositions or delivery vehicles are targetedto endothelial cells, sites of vascular inflammation, blood clots,thromboses, or combinations thereof. For example, vascular inflammationis discussed in Inoue, et al., JACC: Cardiovascular Interventions,4(10):1057-1066 (2011), which is specifically incorporated by referencein its entirety, and discusses various factors, proteins, cytokines, andcell types that are localized to sites of vascular inflammation andtherefore can serve as targets for the targeting signals used with thecompositions and delivery vehicles disclosed herein. Components of clotsand thromboses may are used as suitable targets. Among these markers ortargets are fibrin, gpIIb/IIIa, tissue factor/VIIA complex, activatedclotting factor Xa, activated clotting factor IXa, and the fibrincondensation product, d-dimer.

In some embodiments, the targeting signal is incorporated into or linkedto a delivery vehicle. For example, if the delivery vehicle is apolymeric particle, the targeting molecules can be coupled directly tothe particle or to an adaptor element such as a fatty acid which isincorporated into the polymer. Ligands may be directly attached to thesurface of polymeric particles via a functional chemical group(carboxylic acids, aldehydes, amines, sulfhydryls and hydroxyls) presenton the surface of the particle and present on the ligand to be attached.Functionality may be introduced post-particle preparation, by directcrosslinking of particles and ligands with homo- or heterobifunctionalcrosslinkers. This procedure may use a suitable chemistry and a class ofcrosslinkers (CDT, EDAC, glutaraldehydes, etc. as discussed in moredetail below) or any other crosslinker that couples ligands to theparticle surface via chemical modification of the particle surface afterpreparation.

Ligands may also be attached to polymeric particles indirectly thoughadaptor elements which interact with the polymeric particle. Adaptorelements may be attached to polymeric particles in at least two ways.The first is during the preparation of the micro- and nanoparticles, forexample, by incorporation of stabilizers with functional chemical groupsduring emulsion preparation of microparticles. For example, adaptorelements, such as fatty acids, hydrophobic or amphiphilic peptides andpolypeptides can be inserted into the particles during emulsionpreparation. In a second embodiment, adaptor elements may be amphiphilicmolecules such as fatty acids or lipids which may be passively adsorbedand adhered to the particle surface, thereby introducing functional endgroups for tethering to ligands. Adaptor elements may associate withmicro- and nanoparticles through a variety of interactions including,but not limited to, hydrophobic interactions, electrostatic interactionsand covalent coupling.

In some embodiments, the targeting signal is or includes a proteintransduction domain, also known as cell penetrating peptides (CPPS).PTDs are known in the art, and include but are not limited to smallregions of proteins that are able to cross a cell membrane in areceptor-independent mechanism (Kabouridis, P., Trends in Biotechnology(11):498-503 (2003)). The two most commonly employed PTDs are derivedfrom TAT (Frankel and Pabo, Cell, December 23; 55(6):1189-93 (1988))protein of HIV and Antennapedia transcription factor from Drosophila,whose PTD is known as Penetratin (Derossi et al., J Biol Chem.269(14):10444-50 (1994)).

The Antennapedia homeodomain is 68 amino acid residues long and containsfour alpha helices. Penetratin is an active domain of this protein whichconsists of a 16 amino acid sequence derived from the third helix ofAntennapedia (SEQ ID NO:3). TAT protein (SEQ ID NO:4) consists of 86amino acids and is involved in the replication of HIV-1. The TAT PTDconsists of an 11 amino acid sequence domain (residues 47 to 57;YGRKKRRQRRR (SEQ ID NO:5) of the parent protein that appears to becritical for uptake. Additionally, the basic domain Tat(49-57) orRKKRRQRRR (SEQ ID NO:6) has been shown to be a PTD.

Several modifications to TAT, including substitutions of Glutatmine toAlanine, i.e., Q→A, have demonstrated an increase in cellular uptakeanywhere from 90% to up to 33 fold in mammalian cells. (Ho et al.,Cancer Res. 61(2):474-7 (2001)) The most efficient uptake of modifiedproteins was revealed by mutagenesis experiments of TAT-PTD, showingthat an 11 arginine stretch was several orders of magnitude moreefficient as an intercellular delivery vehicle. Thus, some embodimentsinclude PTDs that are cationic or amphipathic. Additionally exemplaryPTDs include but are not limited to poly-Arg—RRRRRRR (SEQ ID NO:7);PTD-5—RRQRRTSKLMKR (SEQ ID NO:8); TransportanGWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:9);KALA—WEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:10); and RQIKIWFQNRRMKWKK(SEQ ID NO:11).

C. Delivery Vehicles

The CTPS1 inhibitors can be administered and taken up into the cells ofa subject with or without the aid of a delivery vehicle. Appropriatedelivery vehicles for the disclosed inhibitors are known in the art andcan be selected to suit the particular inhibitor. For example, if theCTPS1 inhibitor is a nucleic acid or vector, the delivery vehicle can bea viral vector, for example a commercially available preparation, suchas an adenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec,Canada). The viral vector delivery can be via a viral system, such as aretroviral vector system which can package a recombinant retroviralgenome (see e.g., Pastan et al., (1988)Proc. Natl. Acad. Sci. U.S.A.85:4486; Miller et al., (1986) Mol. Cell. Biol. 6:2895). The recombinantretrovirus can then be used to infect and thereby deliver to theinfected cells nucleic acid encoding the CTPS1 inhibitor. The exactmethod of introducing the altered nucleic acid into mammalian cells is,of course, not limited to the use of retroviral vectors. Othertechniques are widely available for this procedure including the use ofadenoviral vectors (Mitani et al., Hum. Gene Ther. 5:941-948 (1994)),adeno-associated viral (AAV) vectors (Goodman et al., Blood 84:1492-1500(1994)), lentiviral vectors (Naidini et al., Science 272:263-267(1996)), pseudotyped retroviral vectors (Agrawal et al., Exper. Hematol.24:738-747 (1996)).

Physical transduction techniques can also be used, such as liposomedelivery and receptor-mediated and other endocytosis mechanisms (see,for example, Schwartzenberger et al., Blood 87:472-478 (1996)). Forexample in some embodiments, the CTPS1 inhibitor is delivered via aliposome. Commercially available liposome preparations such asLIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.),SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (PromegaBiotec, Inc., Madison, Wis.), as well as other liposomes developedaccording to procedures standard in the art are well known. In addition,the disclosed nucleic acid or vector can be delivered in vivo byelectroporation, the technology for which is available from Genetronics,Inc. (San Diego, Calif.) as well as by means of a SONOPORATION machine(ImaRx Pharmaceutical Corp., Tucson, Ariz.). This disclosed compositionsand methods can be used in conjunction with any of these or othercommonly used gene transfer methods.

In some embodiments, the delivery vehicle is incorporated into orencapsulated by a nanoparticle, microparticle, micelle, syntheticlipoprotein particle, or carbon nanotube. For example, the compositionscan be incorporated into a vehicle such as polymeric microparticleswhich provide controlled release of the CTPS1 inhibitor. In someembodiments, release of the drug(s) is controlled by diffusion of theCTPS1 inhibitor out of the microparticles and/or degradation of thepolymeric particles by hydrolysis and/or enzymatic degradation. Suitablepolymers include ethylcellulose and other natural or synthetic cellulosederivatives. Polymers which are slowly soluble and form a gel in anaqueous environment, such as hydroxypropyl methylcellulose orpolyethylene oxide may also be suitable as materials for drug containingmicroparticles. Other polymers include, but are not limited to,polyanhydrides, poly (ester anhydrides), polyhydroxy acids, such aspolylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide)(PLGA), poly-3-hydroxybut rate (PHB) and copolymers thereof,poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactoneand copolymers thereof, and combinations thereof.

The CTPS1 inhibitor can be incorporated into prepared from materialswhich are insoluble in aqueous solution or slowly soluble in aqueoussolution, but are capable of degrading within the GI tract by meansincluding enzymatic degradation, surfactant action of bile acids, and/ormechanical erosion. As used herein, the term “slowly soluble in water”refers to materials that are not dissolved in water within a period of30 minutes. Preferred examples include fats, fatty substances, waxes,waxlike substances and mixtures thereof. Suitable fats and fattysubstances include fatty alcohols (such as lauryl, myristyl stearyl,cetyl or cetostearyl alcohol), fatty acids and derivatives, including,but not limited to, fatty acid esters, fatty acid glycerides (mono-, di-and tri-glycerides), and hydrogenated fats. Specific examples include,but are not limited to hydrogenated vegetable oil, hydrogenatedcottonseed oil, hydrogenated castor oil, hydrogenated oils availableunder the trade name Sterotex®, stearic acid, cocoa butter, and stearylalcohol. Suitable waxes and wax-like materials include natural orsynthetic waxes, hydrocarbons, and normal waxes.

Specific examples of waxes include beeswax, glycowax, castor wax,carnauba wax, paraffins and candelilla wax. As used herein, a wax-likematerial is defined as any material which is normally solid at roomtemperature and has a melting point of from about 30 to 300° C.

Micro and nanoparticles designed to deliver cargo such as a drugs andimaging reagents to the vasculature, to vascular smooth muscle cells, orsites or clots or thrombosis are known art. See, for example, Wickline,et al., Arteriosclerosis, Thrombosis, and Vascular Biology, 26:435-441(2006), published online (December 2005), and U.S. Published ApplicationNos. 2002/0168320, 2003/0086867, 2003/0129136, 2004/0058951,2004/0115192, 2006/0147380, 2006/0239919, 2007/0140965, 2007/0202040,2007/0258908, 2008/0175792, 2008/0247943, and 2013/0064765, each ofwhich are specifically incorporated by reference herein in its entirety.

For example, perfluorocarbon nanoparticles, previously considered asartificial blood substitutes, have been developed into a platformtechnology for molecular imaging and targeted drug delivery, i.e., aso-called “theranostic” technology. These lipid-encapsulated particles,which are nominally 250 nm in diameter, can be administeredintravenously and are typically constrained by size to the intactvasculature.

Preferred vehicles for delivery of nucleoside analogs, including, butnot limited to, polymer-based nanoparticles and polyplex nanogelformulations are also known in the art. See, for example, Hillaireau, etal., J. Nanosci. Nanotechnol., 6(9-10):2608-17 (2006), Vinogradov, etal, J. Control Release, 107(1): 143-57 (2005), and Vinogradov, ExpertOpin Drug Deliv. 4(1): 5-17 (2007), each of which is specificallyincorporated by reference in its entirety.

D. Formulations

Pharmaceutical compositions including one or more CTPS1 inhibitors arealso disclosed.

1. Pharmaceutical Compositions

Pharmaceutical compositions including a CTPS1 inhibitor, and optionallya targeting moiety, a delivery vehicle, or a combination thereof areprovided. Pharmaceutical compositions can be for administration byparenteral (intramuscular, intraperitoneal, intravenous (IV) orsubcutaneous injection), transdermal (either passively or usingiontophoresis or electroporation), or transmucosal (nasal, vaginal,rectal, or sublingual) routes of administration or using bioerodibleinserts and can be formulated in dosage forms appropriate for each routeof administration.

In certain embodiments, the compositions are administered locally, forexample by injection directly into a site to be treated. In someembodiments, the compositions are injected, topically applied, orotherwise administered directly into the vasculature onto vasculartissue at or adjacent to a site of injury, surgery, or implantation. Forexample, in embodiments, the compositions are topically applied tovascular tissue that is exposed, during a surgical or implantation, ortransplantation procedure. Typically, local administration causes anincreased localized concentration of the compositions which is greaterthan that which can be achieved by systemic administration.

a. Formulations for Parenteral Administration

Compositions including those containing a CTPS1 inhibitor, andoptionally a targeting moiety, a delivery vehicle, or a combinationthereof are administered in an aqueous solution, by parenteralinjection. The formulation may also be in the form of a suspension oremulsion. In general, pharmaceutical compositions are provided includingeffective amounts of the CTPS1 inhibitor and optionally includepharmaceutically acceptable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or carriers. Such compositions includediluents sterile water, buffered saline of various buffer content (e.g.,Tris-HCl, acetate, phosphate), pH and ionic strength; and optionally,additives such as detergents and solubilizing agents (e.g., TWEEN® 20,TWEEN® 80 also referred to as polysorbate 20 or 80), anti-oxidants(e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g.,Thimersol, benzyl alcohol) and bulking substances (e.g., lactose,mannitol). Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Theformulations may be lyophilized and redissolved/resuspended immediatelybefore use. The formulation may be sterilized by, for example,filtration through a bacteria retaining filter, by incorporatingsterilizing agents into the compositions, by irradiating thecompositions, or by heating the compositions.

b. Oral Formulations

Oral formulations may be in the form of chewing gum, gel strips, tabletsor lozenges. Encapsulating substances for the preparation ofenteric-coated oral formulations include cellulose acetate phthalate,polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate andmethacrylic acid ester copolymers. Solid oral formulations such ascapsules or tablets are preferred. Elixirs and syrups also are wellknown oral formulations. The components of aerosol formulations includesolubilized active ingredients, antioxidants, solvent blends andpropellants for solution formulations, and micronized and suspendedactive ingredients, dispersing agents and propellants for suspensionformulations. The oral, aerosol and nasal formulations of the inventioncan be distinguished from injectable preparations of the prior artbecause such formulations may be nonaseptic, whereas injectablepreparations must be aseptic.

c. Formulations for Topical Administration

The CTPS1 inhibitor, and optionally a targeting moiety, a deliveryvehicle, or a combination thereof can be applied topically. Topicaladministration can include application to the lungs, nasal, oral(sublingual, buccal), vaginal, or rectal mucosa.

Compositions can be delivered to the lungs while inhaling and traverseacross the lung epithelial lining to the blood stream when deliveredeither as an aerosol or spray dried particles having an aerodynamicdiameter of less than about 5 microns.

A wide range of mechanical devices designed for pulmonary delivery oftherapeutic products can be used, including but not limited tonebulizers, metered dose inhalers, and powder inhalers, all of which arefamiliar to those skilled in the art. Some specific examples ofcommercially available devices are the Ultravent® nebulizer(Mallinckrodt Inc., St. Louis, Mo.); the Acorn® II nebulizer (MarquestMedical Products, Englewood, Co.); the Ventolin® metered dose inhaler(Glaxo Inc., Research Triangle Park, N.C.); and the Spinhaler® powderinhaler (Fisons Corp., Bedford, Mass.). Nektar, Alkermes and Mannkindall have inhalable insulin powder preparations approved or in clinicaltrials where the technology could be applied to the formulationsdescribed herein.

Formulations for administration to the mucosa will typically be spraydried drug particles, which may be incorporated into a tablet, gel,capsule, suspension or emulsion. Standard pharmaceutical excipients areavailable from any formulator.

Transdermal formulations may also be prepared. These will typically beointments, lotions, sprays, or patches, all of which can be preparedusing standard technology. Transdermal formulations can includepenetration enhancers.

2. Effective Amounts

In some in vivo approaches, the compositions are administered to asubject in a therapeutically effective amount. As used herein the term“effective amount” or “therapeutically effective amount” means a dosagesufficient to treat, inhibit, or alleviate one or more symptoms of thedisorder being treated or to otherwise provide a desired pharmacologicand/or physiologic effect.

The amount of composition administered to the subject is typicallyeffective to reduce or inhibit proliferation of smooth muscle cells,particularly, vascular smooth muscle cells. In some embodiments, theamount is effective to reduce or inhibit proliferation of smooth musclecells to a greater extent than reducing or inhibiting proliferation ofendothelial cells. In some embodiments, the amount is effective toreduce or inhibit proliferation of smooth muscle cells withoutsubstantially reducing or inhibiting proliferation of endothelial cells.

In some embodiments the amount is effective to reduce or inhibitproliferation of both smooth muscle cells and endothelial cells in theabsence of cytidine. As discussed in more detail below, cytidine can beco-administered to the subject with the CPTS1 inhibitor to induce theCTP synthesis salvage pathway and rescue proliferation in theendothelial cells. Accordingly, when co-administered with cytidine,proliferation of smooth muscles cells is inhibited, but endothelialcells can continue to proliferate even at high doses of CPTS1 inhibitor.Cytidine is typically administered in such a way that it can contact theendothelial cells. For example, cytidine can be injected are infusedinto the blood stream.

The composition can also be administered in an amount effective toreduce or inhibit migration, particularly PDGF-BB-induced SMC migration,of smooth muscle cells.

In the most preferred embodiments, the composition is administered in anamount to prevent, reduce, or inhibit neointima. For example, the amountcan be effective to reduce proliferation, migration, or a combinationthereof of smooth muscle cells from the media layer into the intima. Theamount can be effective to prevent, reduce, or inhibit the rise orappearance of fused intima and media. The amount can be effective toreduce proliferation, migration, or a combination thereof of intimasmooth muscle cells.

Preferably, the composition is administered in an amount that iseffective to prevent, reduce, or inhibit neointima without preventingre-endothelialization at the site of a vascular injury. In someembodiments, the compositions promote re-endothelialization aftervascular injury.

The precise dosage will vary according to a variety of factors such assubject-dependent variables (e.g., age, immune system health, etc.), thedisease, and the treatment being effected. As further studies areconducted, information will emerge regarding appropriate dosage levelsfor treatment of various conditions in various patients, and theordinary skilled worker, considering the therapeutic context, age, andgeneral health of the recipient, will be able to ascertain properdosing. The selected dosage depends upon the desired therapeutic effect,on the route of administration, and on the duration of the treatmentdesired. Generally dosage levels of 0.001 to 10 mg/kg of body weightdaily are administered to mammals. Generally, for intravenous injectionor infusion, dosage may be lower.

Preferred dosages for some of the CTPS1 inhibitors disclosed herein areprovided above or known in the art. It is believed, however, that thedosage for reducing neointima formation can be lower than the dosagesthat are typically administered in the art for treatment of infectionsor cancer.

III. Devices and Grafts

The compositions disclosed herein can be coated onto or incorporatedinto medical devices, or to pre-treat implantable vascular grafts exvivo.

A. Devices

In some embodiments, a composition including one or more CTPS1inhibitors is coated onto, or incorporate into, a medical device toreduce or inhibit neointima formation in a subject. The device can be adevice that is inserted into the subject transiently, or a device thatis implanted permanently. In some embodiments, the device is surgicaldevice.

Examples of medical devices include, but are not limited to, needles,cannulas, catheters, shunts, balloons, and implants such as stents andvalves.

In some embodiments, the CTPS1 inhibitor or pharmaceutical compositioncan be formulated to permit its incorporation onto the medical device,which can apply the inhibitor directly to the site to prevent or treatconditions such restenosis or another vascular proliferation disorder.

In some embodiments the CTPS1 inhibitor or pharmaceutical compositionthereof is formulated by including it within a coating on the medicaldevice. There are various coatings that can be utilized such as, forexample, polymer coatings that can release the inhibitor over aprescribed time period. The inhibitor, or a pharmaceutical compositionthereof, can be embedded directly within the medical device. In someembodiments, the CTPS1 inhibitor is coated onto or within the device ina delivery vehicle such as a microparticle or liposome that facilitatesits release and delivery. In some embodiments, the CTPS1 inhibitor ismiscible in the coating.

In some embodiments, the medical device is a vascular implant such as astent. Stents are utilized in medicine to prevent or eliminate vascularrestrictions. The implants may be inserted into a restricted vesselwhereby the restricted vessel is widened. The experience with suchvascular implants indicates that excessive growth of the adjacent cellsresults again in a restriction of the vessel particularly at the ends ofthe implants which results in reduced effectiveness of the implants. Ifa vascular implant is inserted into a human artery for the eliminationof an arteriosclerotic stenosis, intimahyperplasia can occur within ayear at the ends of the vascular implant and results in renewedstenosis.

Accordingly, in some embodiments, the stents are coated or loaded with acomposition including a CPTS1 inhibitor and optionally a targetingsignal, a delivery vehicle, or a combination thereof. Many stents arecommercially available or otherwise know in the art.

Stents can be formed, i.e., etched or cut, from a thin tube of suitablematerial, or from a thin plate of suitable material and rolled into atube. Suitable materials for the stent include but are not limited tostainless steel, iridium, platinum, gold, tungsten, tantalum, palladium,silver, niobium, zirconium, aluminum, copper, indium, ruthenium,molybdenum, niobium, tin, cobalt, nickel, zinc, iron, gallium,manganese, chromium, titanium, aluminum, vanadium, and carbon, as wellas combinations, alloys, and/or laminations thereof. For example, thestent may be formed from a cobalt alloy, such as L605 or MP35N®, Nitinol(nickel-titanium shape memory alloy), ABI (palladium-silver alloy),Elgiloy® (cobalt-chromium-nickel alloy), etc. It is also contemplatedthat the stent may be formed from two or more materials that arelaminated together, such as tantalum that is laminated with MP35N®. Thestents may also be formed from wires having concentric layers ofdifferent metals, alloys, or other materials. Embodiments of the stentmay also be formed from hollow tubes, or tubes that have been filledwith other materials. The aforementioned materials and laminations areintended to be examples and are not intended to be limiting in any way.

Stents can also be composed of and/or coated with one or more degradablematerials. For example, absorbable materials to make stents and stentcoatings are described in U.S. Pat. Nos. 5,059,211 and 5,306,286; U.S.Pat. No. 5,935,506 describes a method to manufacture an absorbable stentfrom poly-3-hydroxybutyrate (P3HB); U.S. Pat. No. 6,045,568 describesabsorbable stents manufactured from knitting yarns of polyactic acid(PLA), polyglycolic acid (PGA), polyglactin (P(GA-co-LA)), polydioxanone(PDS), polyglyconate (a block co-polymer of glycolic acid andtrimethylene carbonate, P(GA-co-TMC)), and a copolymer of glycolic acidor lactic acid with ε-caprolactone (P(GA-co-CL) or P(LA-co-CL)); andLaaksovirta et al., describes a self-expandable, biodegradable,self-reinforced stent from P(GA-co-LA) for use in urethral applications(J Urol. 2003 August; 170(2 Pt 1):468-71). The use of polyanhydride andpolyorthoester polymers to manufacture absorbable stents is described byTanguay, J. F. et al., Cardiology Clinics, 12:699-713 (1994). WO98/51812 to Williams et al. discloses methods to remove pyrogens frompolyhydroxyalkanoates, and the fabrication of stents with thesedepyrogenated materials and WO 99/32536 to Martin et al. and WO 00/56376to Williams et al. disclose methods to prepare polyhydroxyalkanoateswith controlled degradation rates, and the fabrication of stents withthese materials. Van der Giessen et al. (Marked Inflammatory Sequelae toImplantation of Biodegradable and Nonbiodegradable Polymers in PorcineCoronary Arteries, Circulation, 94:1690-1697 (1996)) evaluated coatingsof a copolymer of glycolic acid and lactic acid (P(GA-co-LA)),polycaprolactone (PCL), poly-3-hydroxybutyrate-co-3-hydroxyvalerate(P(3HB-co-3HV), a polyorthoester, and a polyethyleneoxide-polybutyleneterephthalate on metal stents, and reported that the coatings inducedmarked inflammatory reactions within the coronary artery. Otherbioresorbable stent materials include iron, magnesium, zinc, and theiralloys.

In some embodiments the stent is composed of two or more bioabsorbablepolymers. In some embodiments, the stent is coated with one or morebioabsorbable polymers. The stent can be composed of and coated with thesame or different polymers. Method of making and coating absorbablestents are described in U.S. Pat. No. 7,618,448. Stents can include twoor more coatings, for example, a base coat and one or more top coatscomposed of the same or different polymers.

In some embodiments, the stent is a drug-eluting stent. Various drugeluting stents that simultaneously deliver a therapeutic substance tothe treatment site while providing artificial radial support to the walltissue are known in the art. Endoluminal devices including stents aresometimes coated on their outer surfaces with a substance such as a drugreleasing agent, growth factor, or the like. Stents have also beendeveloped having a hollow tubular structure with holes or ports cutthrough the sidewall to allow drug elution from a central lumen.Although the hollow nature of the stent allows the central lumen to beloaded with a drug solution that is delivered via the ports or holes inthe sidewall of the stent, the hollow tubular structure may not havesuitable mechanical strength to provide adequate scaffolding in thevessel.

In some embodiments, the devices are also coated or impregnated with aCTPS1 inhibitor and one or more additional therapeutic agents,including, but not limited to, antiplatelet agents, anticoagulantagents, anti-inflammatory agents antimicrobial agents, antimetabolicagents, additional anti-neointima agents, additional antiproliferativeagents, immunomodulators, antiproliferative agents, agents that affectmigration and extracellular matrix production, agents that affectplatelet deposition or formation of thrombosis, and agents that promotevascular healing and re-endothelialization, such as those and othersdescribed in Tanguay et al. Cardiology Clinics, 12:699-713 (1994), J. E.Sousa, et al., Circulation, 107 (2003) 2274 (Part I), 2283 (Part II),Salu, et al., Acta Cardiol, 59 (2004) 51.

Examples of antithrombin agents include, but are not limited to, Heparin(including low molecular heparin), R-Hirudin, Hirulog, Argatroban,Efegatran, Tick anticoagulant peptide, and PPACK.

Examples of antiproliferative agents include, but are not limited to,Paclitaxel (Taxol), QP-2 Vincristin, Methotrexat, Angiopeptin,Mitomycin, BCP 678, Antisense c-myc, ABT 578, Actinomycin-D, RestenASE,1-Chlor-deoxyadenosin, PCNA Ribozym, and Celecoxib.

Examples of anti-restenosis agents include, but are not limited to,immunomodulators such as Sirolimus (Rapamycin), Tacrolimus, Biorest,Mizoribin, Cyclosporin, Interferon-γ 1b, Leflunomid, Tranilast,Corticosteroide, Mycophenolic acid and Biphosphonate.

Examples of anti-migratory agents and extracellular matrix modulatorsinclude, but are not limited to Halofuginone,Propyl-hydroxylase-Inhibitors, C-Proteinase-Inhibitors, MMP-Inhibitors,Batimastat, Probucol.

Examples of antiplatelet agents include, but are not limited to,heparin.

Examples of wound healing agents and endothelialization promotersinclude vascular epithelial growth factor (“VEGF”), 17β-Estradiol,Tkase-Inhibitors, BCP 671, Statins, nitric oxide (“NO”)-Donors, andendothelial progenitor cell (“EPC”)-antibodies.

Besides coronary applications, drugs and active agents may beincorporated into the stent or stent coating for other indications. Forexample, in urological applications, antibiotic agents may beincorporated into the stent or stent coating for the prevention ofinfection. In gastroenterological and urological applications, activeagents may be incorporated into the stent or stent coating for the localtreatment of carcinoma.

It may also be advantageous to incorporate in or on the stent a contrastagent, radiopaque markers, or other additives to allow the stent to beimaged in vivo for tracking, positioning, and other purposes. Suchadditives could be added to the absorbable composition used to make thestent or stent coating, or absorbed into, melted onto, or sprayed ontothe surface of part or all of the stent. Preferred additives for thispurpose include silver, iodine and iodine labeled compounds, bariumsulfate, gadolinium oxide, bismuth derivatives, zirconium dioxide,cadmium, tungsten, gold tantalum, bismuth, platinum, iridium, andrhodium. These additives may be, but are not limited to, mircro- ornano-sized particles or nano particles. Radio-opacity may be determinedby fluoroscopy or by x-ray analysis.

A CTPS1 inhibitor and one or more additional agents can be incorporatedinto the stent, either by loading the agent(s) into the absorbablematerial prior to processing, and/or coating the surface of the stentwith the agent(s). The rate of release of agent may be controlled by anumber of methods including varying the following the ratio of theabsorbable material to the agent, the molecular weight of the absorbablematerial, the composition of the agent, the composition of theabsorbable polymer, the coating thickness, the number of coating layersand their relative thicknesses, and/or the agent concentration. Topcoats of polymers and other materials, including absorbable polymers,may also be applied to active agent coatings to control the rate ofrelease. For example, P4HB can be applied as a top coat on a metallicstent coated with P4HB including an active agent to retard the releaseof the active agent.

Exemplary stents that can be used with the compositions and methodsdisclosed herein include, but are not limited to, those described inU.S. Pat. Nos. 5,891,108, 6,918,929, 6,923,828, 6,945,992, 6,986,785,7,060,090, 7,144,419, 7,163,555, 7,323,008, 7,651,527, 7,655,034,7,678,141, 7,744,645, 7,942,917, 8,001,925, 8,001,925, 8,034,099,8,048,149, 8,066,760, 8,100,960, 8,157,855, 8,172,893, 8,182,524,8,187,284, 8,187,322, 8,197,528, 8,206,432, 8,221,490, 8,231,669,8,236,044, 8,252,048, 8,252,065, 8,257,425, 8,257,431, 8,292,945,8,298,278, 8,298,280, 8,348,991, 8,348,992, 8,348,993, 8,353,952,8,359,998, 8,361,140, 8,372,134, 8,372,138, 8,377,112, 8,388,676,8,398,695, 8,414,637, 8,414,639, and 8,414,656.

B. Grafts

The compositions can also be used to pre-treat vascular grafts ex vivoprior to implantation in a subject. The compositions including one ormore CTPS1 inhibitor and optionally a targeting single, a deliveryvehicle or a combination thereof can be applied to the tissue by methodsto insure that they adhere and are distributed throughout the tissue inoptimal locations for drug treatment. In some embodiments, the CTPS1 isdelivered using a nanoparticle or microparticle that is designed theyadhere to the regions of interest, to carry sufficient drug load toprovide local treatment for prolonged periods, to release the drug loadat the proper rate, to penetrate into tissue to an optimal extent, or acombination thereof. The surgeon can then apply a long-term, local drugregimen at the same time the tissue or organ is placed in the patient.

For example, the amount of inhibitor present on the graft tissue and thepenetration of the inhibitor throughout the tissue can be adjusted, bychanging the formulation or delivery vehicle. In this way the amount ofdrug locally release at the site of implantation can be carefullycontrolled. Typically, the CTPS1 inhibitor, or a delivery vehiclecarrying the inhibitor is contacted with the graft material ex vivo. Thecontacting can occur in the absence or presence of mild agitation, orother methods known in the art to insure that inhibitor attaches to orpenetrates the graft tissue. Agitation may be accomplished for example,by incubation on an orbital shaker, or by vertical rotation, such as byincubation in a vertical carousel of a hybridization oven. Theincubation protocol can be varied to affect the positioning of theparticles on the graft. The amount and localization of attachment ofdelivery vehicles such as particles to the graft can also be varied byvarying the type and density of attachment and targeting ligands, suchas those described above, presented on the vehicle. Compositions andmethods for delivering drugs to vascular grafts ex vivo are discussed inU.S. Published Application Nos. 2006/0002971, 2010/0151436, and U.S.Pat. No. 7,534,448.

The compositions and methods can be used to locally deliveranti-restenotic agents to grafts with, or without the requirement forfurther invasive procedures, such as placement of a stent. Examples ofvascular grafts, include, but not limited to, bypass grafts andarteriovenous grafts. The grafts can be autologous, for example,saphenous vein or radial artery; preserved autologous, for examplecryopreserved vein; allogeneic; xenogenic; or synthetic, for example,woven polyester, polyurethane (LYCRA®), polytetraflouroethylene (PTFE),GORE-TEX®, or polyethylene terephthalate (DACRON®). For example,arterial homograft using internal mammary, radial or hypogastricarteries are examples of useful and durable vascular conduits.

Exemplary graft procedures are discussed below.

1. Bypass Graft

A common form of bypass surgery involves resecting the saphenous veinfrom the leg for autotransplantation to the coronary artery. In asignificant number of cases these grafts fail, largely due to restenosiscaused by neointimal hyperplasia. The methods described herein can beused to deliver one or more CTPS1 inhibitors locally and in a controlledfashion, to the autologous graft. The application of the inhibitor canbe done during surgery. After resection of the saphenous vein the tissuecan be (and is often for hours) suspended in saline during chest openingand preparation for graft implantation. Compositions including one ormore CTPS1 inhibitors and optional a targeting signal, delivery vehicleor combination thereof can be incubated with the saphenous vein duringthis time period.

2. Arteriovenous Graft

End stage renal disease is increasing in the United States. Morbidity ofhemodialysis access remains a major quality of life issue for patients;it also represents a significant cost to society. A native arteriovenousfistula (AVF) remains the conduit of choice to provide access forhemodialysis and provides superior results when compared with otheroptions such as a prosthetic AVG. Unfortunately each individual islimited in the number of native AVF that can be created due to thelimited number of suitable sites and vessels. Access sites are limitedas patients with end stage renal disease usually have severecomorbidity, requiring extensive venipuncture for diagnosis and therapyfor life. In patients who have exhausted all options for primary AVF,new access sites must use AV grafts; these grafts are susceptible torestenosis by neointimal hyperplasia limiting their effectiveness.Incubation of a composition including one or more CTPS1 inhibitors, andoptionally including a targeting signal, a delivery vehicle, or acombination thereof onto the graft can be done at the time of surgery.

IV. Methods of Reducing or Preventing Neointima Formation

Methods of preventing, reducing, or inhibiting neointima formation aredisclosed. The methods can include administering a subject an effectiveamount of a composition including a CTPS1 inhibitor to prevent, reduce,or inhibit neointima formation in the subject; pretreating a medicaldevice or vascular graft with an effective amount a compositionincluding a CTPS1 inhibitor to prevent, reduce, or inhibit neointimaformation following insertion or implantation of the device or graftinto the subject; or a combination thereof.

The methods typically reduce or inhibit proliferation of smooth musclecells, particularly, vascular smooth muscle cells, compared to acontrol. In some embodiments, the methods reduce or inhibitproliferation of smooth muscle cells to a greater extent thanendothelial cells, or without reducing or inhibiting proliferation ofendothelial cells.

Proliferating smooth muscle cells secrete factors that can reduce orinhibit the proliferation of endothelial cells which can reduce or delayre-endothelialization. Therefore, in some embodiments, the methods donot reduce, or even enhance or promote re-endothelialization. Themethods can reduce proliferation, migration, or a combination thereof ofsmooth muscle cells from the media layer into the intima. In the mostpreferred embodiments, the methods prevent, reduce, or inhibit the riseor appearance of fused intima and media.

Suitable controls are known in the art, and include, for example,untreated cells, or an untreated subject. In some embodiments, thecontrol is untreated tissue for the subject that is treated, or from anuntreated subject. Preferably the cells or tissue of the control are thederived from the same tissue as the treated cells or tissue. In someembodiments, an untreated control subject suffers from the samecondition as the treated subject, for example, injured or enthothelialcell-denuded vasculature.

A. Methods of Treatment

Inhibition of CTPS1 can be a pathway that can be therapeuticallytargeted through either local or systemic delivery. In some embodiments,the compositions are administered systemically and targeted to thevasculature using a targeting signal such as those discussed above. Insome embodiments, the compositions are administered directly to thelocal site of vascular injury.

It is believed that CTPS1 is an early target in the restenosis processand as such, early pharmacological intervention can preclude chronictherapy and any potentially adverse side effects associated with chronictherapy. For example, the compositions disclosed herein can reduce orprevent neointima formation, but allow re-endothelialization to occur.In some embodiments, the therapy can be discontinued oncere-endothelialization has occurred.

In some embodiments, the compositions are coated onto or incorporatedinto devices or grafts as discussed above.

B. Combination Therapies

The compositions, devices, and grafts disclosed herein can be used incombination with one or more additional therapeutic agents. The term“combination” or “combined” is used to refer to either concomitant,simultaneous, or sequential administration of two or more agents.Therefore, the combinations can be administered either concomitantly(e.g., as an admixture), separately but simultaneously (e.g., viaseparate intravenous lines into the same subject), or sequentially(e.g., one of the compounds or agents is given first followed by thesecond). The additional therapeutic agents can be administered locallyor systemically to the subject, or coated or incorporated onto, or intoa device or graft.

The additional therapeutic agents are other anti-neointima agents,chemotherapeutic agents, antibodies, antibiotics, antivirals, steroidaland non-steroidal anti-inflammatories, conventional immunotherapeuticagents, immunosuppressants, cytokines, chemokines and/or growth factors.For example, in some embodiments, the CTPS1 inhibitor is combined withother agents such as, including, but not limited to, Paclitaxel,Taxotere, other taxoid compounds, other anti-proliferative agents suchas Methotrexate, anthracyclines such as doxorubicin, immunosuppressiveagents such as Everolimus and Serolimus, and other rapamycin andrapamycin derivatives.

The additional therapeutic agents can be other anti-proliferatives oranti-migrations agents designed for treating or preventing neointimaformation or restenosis. For example, in some embodiments the additionaltherapeutic agent is N-3,4-trihydroxybenzamide or a pharmaceuticallyacceptable salt or ester thereof, didox, imidate, or hydroxyurea asdescribed in U.S. Pat. No. 8,029,815.

As discussed in the Examples below, cytidine can be used to induce theCTP synthesis salvage pathway and rescue proliferation in theendothelial cells when used in combination with a high dose of CTPS1inhibitor. Therefore, in some embodiments, a combination therapyincludes co-administration of cytidine or a cytidine analog such asthose disclosed in WO 2009/058394. In a preferred embodiment, thecytidine or cytidine analog is administered into the subject's bloodstream (e.g., by injection or infusion) where it will contact theluminal surface of the endothelial cells.

C. Diseases to Be Treated

The disclosed CTSP1 inhibitors have a wide variety of uses, for example,they can be used to treat or prevent restenosis or other vascularproliferative disorders following injury or various surgical procedures.Vascular trauma, such as the trauma associated with percutaneoustransluminal coronary angioplasty (PTCA), typically involves a cascadeof molecular and cellular events occurring within the vessel wallinvolving the release of a variety of vasoactive, thrombogenic, andmitogenic factors (Bauters and Isner, Prog Cardiovasc Dis 40:107-116(1997); Libby and Tanaka, Prog Cardiovasc Dis 40:97-106 (1997);Goldschmidt-Clermont and Moldovan, Gene Expr 7:255-260 (1999)). Withinthis cascade, several mechanisms contribute to restenosis includingelastic recoil, thrombosis, smooth muscle cell migration/proliferationand matrix formation. The result of these vascular events is intimalhyperplasia, whereby vascular smooth muscle cells (VSMC's) migrate fromthe media to the intima, proliferate, and consequently form theneointima. During this proliferative response, SMCs undergo a phenotypicmodulation from a contractile to a synthetic phenotype (differentiation)(Epstein et al., Circulation 84:778-787 (1991); Noda-Heiny and Sobel, AmJ Physiol 268:C1195-1201 (1995); Ueda et al., Coron Artery Dis 6:71-81(1995); Farb, et al., Circulation 105:2974-2980 (2002); Indolfi, et al.,Trends Cardiovasc Med 13:142-148 (2003)).

Accordingly, the disclosed compositions, devices, or grafts can beadministered to a subject to reduce or inhibit smooth muscle cellproliferation, migration, and a combination thereof in an amounteffective to reduce or inhibit neointima formation and thereby treat orprevent restenosis and other vascular proliferation disorders in thesubject. A subject can have restenosis or other vascular proliferationdisorders, or be identified as being at risk for restenosis or othervascular proliferation disorders, for example subjects who haveundergone, are undergoing, or will undergo a vascular trauma,angioplasty, surgery, or transplantation arteriopathy, etc.

1. Vascular Trauma

In some embodiments, the subject has undergone, is undergoing, or willundergo a vascular trauma. Vascular trauma include those associated withmedical interventions, such as surgery or angioplasty, also well as bothblunt and penetrating injuries including, but not limited to,lacerations, puncture wounds, crush injuries, gunshot wounds, knifewounds, occupational injuries, falls, and motor vehicle accidents.

2. Surgery

In some embodiments, the subject has undergone, is undergoing, or willundergo a surgery. Surgeries can include invasive, a minimally invasive,or percutaneous surgery. For example, in some embodiments the subject ishaving surgery to treat or repair abdominal aortic aneurysm, carotidstenosis, varicose veins, peripheral arterial occlusive disease, acutelimb ischemia, or aortic dissection. Common vascular surgeries include,but is not limited to, open abdominal aortic aneurysm repair,endovascular aneurysm repair (EVAR), carotid endarterectomy, carotidstenting, vein stripping, sclerotherapy and foam sclerotherapy,endovenous laser treatment, radiofrequency vein ablation, ambulatoryphlebectomy, angioplasty with/out stenting, bypass surgeryendarterectomy atherectomy, balloon embolectomy, thrombectomy, bypasssurgery, open repair, thoracic endovascular aneurysm repair (TEVAR).

3. Angioplasty

In some embodiments, the subject has undergone, is undergoing, or willundergo angioplasty. Angioplasty is the technique of mechanicallywidening narrowed or obstructed arteries, such as those obstructed as aresult of atherosclerosis. Generally, angioplasty includes insertinginto a subject's vasculature an empty and collapsed balloon on a guidewire, known as a balloon catheter, which is passed into the narrowedlocations and then inflated to a fixed size. The balloon forcesexpansion of the inner white blood cell/clot plaque deposits and thesurrounding muscular wall, opening up the blood vessel for improvedflow, and the balloon is then deflated and withdrawn. A stent may or maynot be inserted at the time of ballooning to ensure the vessel remainsopen. Angioplasty includes peripheral angioplasty (i.e., blood vesselsoutside the coronary arteries, such as in the abdomen, or legs),coronary angioplasty, renal artery angioplasty, carotid angioplasty, andcerebral arteries angioplasty.

In some embodiments, the subject has undergone, is undergoing, or willundergo percutaneous transluminal coronary angioplasty (PTCA). The useof PTCA has greatly reduced the number of fatalities in patients whosuffer myocardial infarction (Fischman, et al., N Engl J Med 331:496-501(1994); Elezi, et al., Circulation 98:1875-1880 (1998); Bennett andO'Sullivan, Pharmacol Ther 91:149-166 (2001)). During PTCA, the arterywalls are expanded by several times their original diameter in anattempt to increase lumen diameter and improve flow. Unfortunately, thistechnique is plagued by a high incidence of vessel renarrowing orrestenosis occurring in 30-40% of patients within 6 months of theprocedure (Anderson et al., J Interv. Cardiol., 6:187-202 (1993);Fischman et al., N Engl J Med, 331:496-501 (1994); Elezi et al.,Circulation 98:1875-1880 (1998); Bennett and O'Sullivan, Pharmacol Ther,91:149-166 (2001); Heckenkamp et al., J Cardiovasc. Surg. (Torino),43:349-357 (2002)).

Prevention of restenosis after successful PTCA remains one of the mostchallenging tasks in the treatment of obstructive coronary arterydisease. Attempts to ameliorate this proliferative response involve theuse coronary stents, which have significantly improved both short termand long term outcome following interventional coronaryrevascularization procedures. Despite a reduction in restenosis ratewith stent deployment, restenosis still occurs in 15-30% of patientswithin 6 months (Fischman et al., N Engl J Med, 331:496-501 (1994);Elezi et al., Circulation, 98:1875-1880 (1998)). This incidence ofin-stent restenosis is expected to increase as coronary stenting isbecoming more frequent and is used in less ideal lesions.

4. Transplant Arteriopathy

In some embodiments, the subject has undergone, is undergoing, or willundergo a transplant. Chronic transplant arteriopathy (CTA) is a majorcause of late allograft loss after heart or kidney transplantation(Taylor, et al., J. Heart Lung Transplant., 24:945-955 (2005), Burke, etal., Transplantation, 60:1413-1417 (1995); Cornell and Colvin, Curr.Opin. Nephrol Hypertens., 14:229-234 (2005)). Therefore, in someembodiments, the disclosed compositions and devices are used to reduce,inhibit, or prevent transplant arteriopathy in a transplant recipient.

5. Vascular Proliferative Disorders

In some embodiments, the subject has a vascular proliferative disorder.Examples of such disorders include, but are not limited to, vascularproliferation involved in atherosclerosis, vascular proliferationfollowing intravascular device implantation, vascular proliferation atthe site of vascular anastomosis as generally occurs followingrevascularization procedure or A-V shunting, vascular proliferationfollowing carotid endarderectomy, and transplant vasculopathy.

EXAMPLES Example 1: CTPS1 is Up-Regulated in Cultured SMCs in Vitro andNeointimal SMCs in Vivo

Materials and Methods

Reagents and Cell Culture

Rat aortic smooth muscle cells (SMCs) were cultured by enzyme-digestionmethod from rat thoracic aorta as described previously (Hofer, et al.,Proc Natl Acad Sci USA., 98:6412-6416 (2001), Marquez, et al., BiochemPharmacol., 41:1821-1829 (1991), (Berg, et al., Eur. J. Biochem.,216:161-167 (1993)). SMC phenotype of the cultured cells was confirmedby the expression of smooth muscle alpha-actin and SM22-alpha.

Endothelial cell C166 was purchased from ATCC and grown at 37° C. in ahumidified atmosphere of 5% CO2 in DMEM (Invitrogen) supplemented with10% FBS.

CTPS1 (sc-131474), PCNA (sc-56), CDK1 (sc-137034) and phospho-CDK1(T161) (sc-101654) antibodies were from Santa Cruz Biotechnology (SantaCruz, Calif.). NME1 (#3345S) and NME2 (SAB1400187) antibodies werepurchased from Cell Signaling (Danvers, Mass.), and Sigma-Aldrich (St.Louis, Mo.), respectively.

Animals

Male Sprague-Dawley rats weighing 450 to 500 g and male FVB mouse (13weeks; mean weight 24 g) were purchased from Harlan. All animals werehoused under conventional conditions in the animal care facilities andreceived humane care in compliance with the Principles of LaboratoryAnimal Care formulated by the National Society for Medical Research andthe Guide for the Care and Use of Laboratory Animals. Animal surgicalprocedures were approved by the Institutional Animal Care and UseCommittee of the University of Georgia.

Rat Carotid Artery Injury Model

Rat carotid artery balloon injury was performed using 2F Fogartyarterial embolectomy balloon catheter (Baxter Edwards Healthcare) asdescribed previously (Tulis, Methods Mol. Med., 139:1-30 (2007)). 7, 14or 60 days later, the balloon-injured arteries were perfused withsaline, fixed with 4% paraformaldehyde, embedded in paraffin, andsectioned. Subsequent morphometric analyses were performed in adouble-blinded manner. Ten sections that were evenly distributed in thevessel segments were collected for analysis. The sections were stainedwith modified hematoxylin and eosin or elastica van Gieson (VG) stainingCross-sectional images were captured with a Nikon microscope (NikonAmerica Inc). The circumference of the lumen, internal elastic lamina,and external elastic lamina were measured using Image-pro Plus Software.For immunohistochemistry

(IHC) staining, sections were rehydrated, blocked with 5% goat serum,permeabilized with 0.01% Triton X-100 in PBS, and incubated with primaryantibodies at 4° C. overnight followed by incubation with horseradishperoxidase-conjugated secondary antibody. The sections werecounterstained with hematoxylin.

Statistical Analysis

Each experiment was repeated for more than three times. All values arepresented as means±SEM. Comparisons of parameters between two groupswere made by t test. Comparisons of parameters among more than twogroups were made by one-way analysis of variance, and comparisons ofdifferent parameters between each group were made by a post hoc analysisusing a Bonferroni test. P values <0.05 were considered to bestatistically significant.

Results

It is established that CTPS is involved in the proliferation of variousdifferent cells (Hofer, et al., Proc. Natl. Acad. Sci., 98:6412 (2001);de Clercq, et al., Biochem. Pharmacol., 41:1821-1829 (1991); Berg, etal., Eur. J. Biochem., 216:161-167 (1993)). Although two different CTPSisotypes (CTPS1 and CTPS2) are identified in mammals, it was discoveredthat CTPS1, but not CTPS2, was markedly up-regulated in SMCs by PDGF-BB(FIGS. 1A-1B, 1E).

PDGF-BB is a well-known SMC mitogen that induces SMC proliferation andneointima formation following vascular injury (Arita, et al.,Circulation., 105:2893-2898 (2002)). PDGF-BB-mediated cell proliferationwas confirmed by the up-regulation of proliferating cell nuclear antigen(PCNA) (FIG. 1B).

SMC proliferation is one of the major events in neointima formationunder pathological conditions (Morishita, et al., Proc. Natl. Acad.Sci., 92:5855 (1995)). CTPS1 expression was examined in rat carotidarteries undergoing vascular remodeling after balloon-injuryImmunohistochemistry of neointima following vascular injury revealedthat CTPS1 was expressed at a low level in the media layer SMCs, but washighly induced in neointimal SMCs. A low level of CTPS1 expression wasalso observed in ECs. Quantitative analysis showed that CTPS1 wassignificantly up-regulated as early as 1 day after vascular injury. Theexpression was gradually increased during neointimal formation (FIGS.1C-1D). PCNA expression correlated with the CTPS1 expression (FIG. 1D).

Example 2: Blocking CTPS Activity Inhibits SMC Proliferation andMigration

Materials and Methods

Reagents

CPEC (compound 375575) was obtained from the Open Chemical Repository ofNational Cancer Institute Developmental Therapeutics Program (DTP).

Construction of Adenoviral Vectors

NME1 and NME2 cDNA were individually subcloned into the XhoI site ofpShuttele-IREShrGFP-1 (Agilent) and was confirmed by sequencing.Adenoviral vectors expressing CTPS1 and NME2 short hairpin RNA (shRNA)(shCTPS1 and shNME2) were constructed and the viruses were purified asdescribed previously (Shi, et al. Arterioscler. Thromb. Vasc. Biol.,31:e19-e26 (2011)). The shRNA sequences were as follows: shCTPS1 topstrand: 5′-CGC GTC GCG CTA GAG CAC TCT GCA TTG GCC ATT AAT TCA AGA GATTAA TGG CCA ATG CAG AGT GCT CTA GCG CTT TTT TCC AAA-3′ (SEQ ID NO:12);shCTPS1 bottom strand: 5′-AGC TTT TGG AAA AAA GCG CTA GAG CAC TCT GCATTG GCC ATT AAT CTC TTG AAT TAA TGG CCA ATG CAG AGT GCT CTA GCG CGA-3′(SEQ ID NO:13); shNME2 top strand: 5′-CGC GTC GAG ATC CAT CTG TGG TTTAAG CCC GAA GAT TCA AGA GAT CTT CGG GCT TAA ACC ACA GAT GGA TCT CTT TTTTCC AAA-3′(SEQ ID NO:14); shNME2 bottom strand: 5′-AGC TTT TGG AAA AAAGAG ATC CAT CTG TGG TTT AAG CCC GAA GAT CTC TTG AAT CTT CGG GCT TAA ACCACA GAT GGA TCT CGA-3′ (SEQ ID NO:15). Green fluorescent protein(GFP)-expressing adenovirus (Ad-GFP) was used as a control.

Wound Healing Assay

Cell migration was evaluated by wound healing assay using the CytoSelectWound Healing Assay Kit (Cell Biolabs). Wound healing inserts were putinto 24-well cell culture plates coated with fibronectin. Cellsuspension (250 μl) was added to either side of the insert and incubatedovernight to form a monolayer. The inserts were then removed to allowthe cells to migrate. Images of wound healing were captured using adissection microscope. Cell migration was quantified by blindlymeasuring the migration distances.

Results

To determine if CTPS1 plays a role in SMC proliferation, CTPS activitywas blocked using cyclopentenyl cytosine (CPEC), an effective andspecific inhibitor for CTPS (Moyer, et al., Cancer Res., 46:3325-3329(1986); Politi, et al., Cancer Chemother. Pharmacol., 36:513-523(1995)). As shown in FIG. 2A, blockade of CTPS activity significantlysuppressed SMC proliferation in a dose dependent manner. CPEC treatmentalso dramatically suppressed PCNA expression (FIG. 2C).

To confirm the specificity of CTPS1 function, CTPS1 expression wasblocked by shRNA. It was discovered that knockdown of CTPS1 (FIG. 2D)effectively suppressed PDGF-BB-induced SMC proliferation (FIG. 2C).CTPS1 shRNA appeared to inhibit vehicle-treated SMC growth as well (FIG.2C). Consistently, CTPS1 knockdown also inhibited PCNA expression (FIG.2D), further demonstrating the important role of CTPS1 in SMCproliferation.

Vascular remodeling following injury is initiated by SMC migration frommedia layer to intima (Nilsson, et al., Arterioscler. Thromb. Vasc.Biol., 17:490-497 (1997)). Wound healing assay showed that blockade ofCTPS1 activity by CPEC inhibited SMC migration in a dose-dependentmanner (FIG. 3A). The importance of CTPS1 in SMC migration was alsoassessed using CTPS1 shRNA. As shown in FIG. 3B, shRNA knockdown ofCTPS1 effectively inhibited PDGF-BB-induced SMC migration, consistentwith the CPEC treatment.

Example 3: Blockade of CTPS1 Activity or Expression does not Induce SMCApoptosis but Impairs Cell Cycle Progression

Materials and Methods

TUNEL Assay

In vivo cell apoptosis was evaluated by detecting DNA fragmentationusing the terminal deoxynucleotidyl transferase (TdT)-mediateddUTP-digoxigenin nick end-labeling method (TUNEL kit, Roche, USA).Apoptotic cells were observed under a fluorescent microscope. In vitrocell apoptosis was measured by Flow Cytometry. Cells were stained withboth Annexin V-FITC (BD Biosciences) and propidium iodide (PI) andanalyzed on a FACSCALIBUR™ (Becton Dickinson). The percentages ofpositive-stained cells were quantified using CELLQUEST™ software (BectonDickinson). Non-stained cells served as controls.

Cell Cycle Flow Cytometry Analysis

1×10⁶ cells were harvested and resuspended in 500 μl of reaction buffercontaining 1 μl of Nuclear-IDTM Red dye (Nuclear-IDTM Red Cell CycleAnalysis Kit, Enzo Life Sciences, USA). After mixing, cells wereincubated in the dark for 15 min. Cell cycle analysis was performed on aFACSCALIBUR™ (Becton Dickinson) and analyzed by the CELLQUEST™ software(Becton Dickinson).

Results

Since CPEC is a chemical with potential cytotoxicity, CPEC inhibited SMCproliferation was tested for a non-specific toxic effect. Apoptosisanalysis using PI/Annexin V double staining and flow cytometry indicatedthat 24 h treatment with 500 nM CPEC (the highest dosage used in thisstudy) did not induce SMC apoptosis, comparable with the treatment withvehicle (FIG. 4A). The serum-starved cells were used as a positivecontrol (PC), which caused a significant cell apoptosis (30.86%) (FIG.4A) (24). However, CPEC treatment or CTPS1 knockdown significantlyblocked the cell cycle progression, as indicated by the accumulation ofcells in G0/G1 phase (FIG. 4B-4C). It appeared that CPEC treatment (FIG.4D) or CTPS1 knockdown by shRNA (FIG. 4E) dramatically inhibited theactivation of cell cycle regulator CDK1 via suppression of its T161phosphorylation site.

Example 4: CTPS1 Plays an Important Role in Injury-Induced VascularRemodeling

Materials and Methods

Adenoviral Gene Transfer

Adenovirus gene transfer was achieved by incubation of 5×10⁹ pfu ofadenovirus in balloon-injured carotid arteries for 20 min as describedpreviously (Dollery, et al., Ann. N. Y. Acad. Sci., 878:742-743 (1999)).

Results

Since CTPS1 played an essential role in SMC proliferation and migrationin vitro and was induced in neointima SMCs following balloon injury, anexperiment was designed to determine if CTPS1 plays an important role ininjury-induced neointima formation in vivo. Rat carotid arteries wereinjured with balloon catheter to induce neointima formation andadministered CPEC via mini-osmotic pumps to block CTPS1 activity. Athick layer of neointima was formed 14 days after injury. However, theneointima was dramatically blocked by a low dosage (1 mg/kg bodyweight/day), and completely blocked by a higher dosage of CPEC (2 mg/kgb.w./day) (FIG. 5A-5B). The PCNA expression was also dramaticallyblocked by the low dose CPEC and completely blocked by the higher doseCPEC as determined by immunohistochemistry.

To confirm the specificity of CTPS1 function, CTPS1 was knockdown usingadenoviral-mediated shRNA delivery in injured arteries. As shown in FIG.5C, CTPS1 shRNA dramatically blocked neointima formation as well as PCNAexpression in neointima SMCs. To determine whether or not CPEC had atoxic effect on neointima SMCs, the in vivo cell apoptosis was detectedby TUNEL assay. Consistent with the in vitro results (FIG. 4A), noapoptotic cells were observed by immunohistochemisty in the vesselsections from CPEC-treated arteries even with the higher dose CPEC (2mg/kg b.w./day). These results demonstrate that CTPS1 is an ideal drugtarget for blocking injury-induced neointima formation/vascularremodeling.

Example 5: Blockade of CTPS1 Activity or Expression Impacted ECsDifferently from SMCs in Vitro and Promoted Re-Endothelialization inVivo

Materials and Methods

Cell Proliferation Assay

Cell proliferation was evaluated with3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay using aTACS MTT Cell Proliferation Assay Kit (Trivegen). The optical density at570 nm was measured.

Mouse Carotid Artery Wire Injury Model

Mice were anesthetized with ketamine hydrochloride (80 mg/kg IP) andxylazine (5 mg/kg IP), a 0.38-mm flexible angioplasty guidewire wasadvanced by 1 cm via a transverse arteriotomy of the external carotidartery, and endothelial denudation of the common carotid artery wasachieved by 3 rotational passes (Werner, et al., Circ. Res., 93:e17-e24(2003)).

Results

Re-endothelialization is an important step toward successful vascularrepair in injury-induced vessel wall remodeling. Current anti-neointimastrategies targeting genes or signaling pathways for SMC proliferationalso block re-endothelialization and cause thrombosis as a side effect(Hofma, et al., Eur. Heart J, 27:166-170 (2006); Hofma, et al., Eur.Heart J, 27:166-170 (2006). It is important, therefore, to identify drugtargets that do not block re-endothelialization. Since CTPS1 played avery important role in neointima formation, experiments were designed todetermine if blocking CTPS1 activity or expression has any effect onECs. CPEC was tested for an effect on EC proliferation and migration invitro. As shown in FIG. 6A, EC proliferation was not affected by lowdosages of CPEC treatments (≤20 nM), in which SMC proliferation wasattenuated, indicating that SMCs are more sensitive than ECs to CPECchallenge. A higher dosage of CPEC (500 nM) significantly blocked theproliferation of both SMCs and ECs, indicating that a certain level ofCTPS activity is required for EC proliferation. Indeed, CTPS1 wasinduced in ECs by different growth stimuli (FIG. 6C). Interestingly,addition of extracellular cytidine restored EC, but not SMC,proliferation that was originally blocked by the high dosage of CPEC(FIG. 6A), indicating that SMCs and ECs may use different pathways toregulate their CTP synthesis, and consequently their proliferation.Since high dosage of CPEC did not have toxic effect on cells (FIG. 4A),ECs were treated with 500 nM of CPEC and it was found that CPEC had noeffect on EC migration although the high dosage of CPEC inhibited ECproliferation (FIG. 6A).

Although cytidine is important for EC proliferation, abundant CTP aloneseemed not to be able to stimulate either EC or SMC proliferation (FIG.6D), probably because the proliferation also requires many other factorsincluding cyclin-dependent kinase activity.

Paclitaxel (Taxus), one of the drugs currently used for coating stentsand treating coronary artery diseases, effectively blocks SMCproliferation and restenosis after angioplasty. However, paclitaxelsuppressed both EC and SMC proliferation at a lower (20 nmol/L) orhigher dosage (500 nmol/L; FIG. 6E), consistent with previous reports.Unlike blockade of CTPS1, paclitaxel-mediated inhibitory effect couldnot be restored by cytidine (FIG. 6E). In addition, ECs seemed to bemore sensitive to paclitaxel than SMCs. These results suggest thatblocking CTPS1 may be a better strategy to block restenosis duringcardiovascular intervention.

To determine if CTPS1 plays a role in re-endothelialization duringvascular remodeling in vivo, CPEC was administered systematically viaosmotic pump into rats with balloon-injury or mice with wire-injury intheir carotid arteries. A weak re-endothelialization was found in thecarotid arteries after 60 days of vascular injury as indicated by theexpression of EC marker CD31, indicating a slow and weak proliferationof ECs during the natural vascular repair process. However, CPECadministration (1 mg/kg/day) induced a strong re-endothelialization 14days after the injury while significantly inhibiting the neointimaformation as compared to saline-treated arteries. Quantification of there-endothelialization areas in Evans blue-stained vessel showed thatCPEC treatment increased the re-endothelialization area to 66% of theinjured vessels compared with 24% in saline-treated vessels (FIG. 6F).Consistently, PCNA was strongly expressed in CD31-positive cells,indicating that CPEC treatment preserved or promoted EC proliferation ininjured vessel.

These results demonstrate that CPEC triggered an alternative pathway,which had preserved or even promoted the re-endothelialization duringvascular repair. The specificity of CPEC effect on CTPS1 activity wasconfirmed by adenovirus-mediated CTPS1 shRNA. Knockdown of CTPS1 inducedre-endothelialization while blocking neointima formation, similar to theeffect observed in CPEC treatment. The re-endothelialization in theinjured vessel segments after CPEC treatment was also observed via EvansBlue staining.

To further establish CTPS function in re-endothelialization aftervascular injury, the endothelial denudation and re-endothelializationwas tested in a different model, i.e., mouse wire-injury model. There-endothelialization was observed in the intact carotid arteries usingEvans Blue staining of the injured areas. Systematic administration ofCPEC was achieved via osmotic pump (1 mg/kg/day). Re-endothelializationin mouse arteries occurred rapidly following injury (FIG. 6B). 5 daysafter injury, re-endothelialization was completed, consistent with theprevious reports (Hagensen, et al., Cardiovasc Res., 93:223-231 (2012)).CEPC treatment did not delay the rapid re-endothelialization.

Importantly, at 3 days after the injury, CPEC-treated arteries appearedto have a larger area that was re-endothelialized compared tosaline-treated arteries (FIG. 6B), indicating that CPEC accelerated there-endothelialization process. Taken together, these data demonstratethat blocking CTPS function or expression promotes re-endothelializationduring injury-induced vascular repair and remodeling.

Example 6: Induction of CTP Synthesis Salvage Pathway Sustains theProliferation of CPEC-Treated ECs and Promotes Re-Endothelialization ofthe Injured Vessel

Materials and Methods

Quantitative RT-PCR (qPCR) and Western Blot

Total RNA was extracted from cells or tissues using Trizol reagent(Invitrogen) and reverse transcribed to cDNA using M-MLV reversetranscriptase (Promega). qPCR was performed on a Stratagene Mx3005 qPCRthermocycler (Agilent Technologies, La Jolla, Calif.). Western blot wasperformed as described previously (Shi, et al., J. Biol. Chem.,287:6860-6867 (2012)).

Results

Since ECs, but not SMCs, used extracellular cytidine to restore cellproliferation in high dose CPEC treatment (FIG. 6A), the alternativepathway that regulates EC proliferation was investigated. CTPSsynthesizes CTP via a so-called “neo-synthesis pathway” using UTP, ATPand glutamine as substrates (Iyengar, et al., Biochem. J., 369:497(2003)). However, there is a “salvage pathway” utilizing cytidine assubstrate when neo-synthesis pathway is in deficiency (Anderson, et al.,Trends Pharmacol. Sci., 18:387-392 (1997); Schimmel, et al., Curr.Cancer Drug Targets., 7:504-509 (2007)). Cytidine is utilized by severalsalvage pathway enzymes, including UCK1/2, CMPK, and nucleosidediphosphate kinase A and B (NME1 and NME2) (Payne, et al., J. Biol.Chem., 260:10242-10247 (1985); Sandeck, et al., PLoS One., 4(8): e6554(2009)).

As shown in FIG. 7A, treatment with CPEC in the presence of cytidinesignificantly induced mRNA expression of all the salvage pathway enzymesin ECs, but not in SMCs, indicating that ECs, but not SMCs, are able touse the salvage pathway when CTPS pathway is blocked. Since NME1/2 playsimilar roles in the salvage pathway as CTPS in the neo-synthesispathway, and the end product of both NMEs and CTPS is CTP, NME1 or NME2are most likely to be responsible for CPEC-induced EC proliferation.CPEC appeared to induce more NEM2 expression than NME1 in proliferativeECs (FIG. 7A). Interestingly, PDGF-BB, VEGF, or CPEC treatment alone didnot upregulate the expression of NME1 or NME2 in ECs. However,combination of CPEC with either one of the growth factors (VEGF or PDGF)in the presence of cytidine dramatically up-regulated the NME especiallythe NME2 expression (FIG. 7B). These data indicate that an elevatedlevel of the salvage pathway activities may be required for ECproliferation when the neo-synthesis pathway is blocked.

Although both NME1 and NME2 are important for the nucleoside diphosphatekinase activity in the salvage CTP synthesis pathway (Postel, et al.,Mol. Cell. Biochem., 329:45-50 (2009)), NME2 overexpression in SMCs(FIG. 7D-7E) displayed a stronger activity than NME1 in utilizingcytidine to restore CPEC-attenuated proliferation (FIG. 7F).Consistently, knockdown of NME2 by adenovirus-expressed shRNAdramatically decreased cytidine-mediated restoration of EC proliferationthat was originally blocked by a high dose CPEC (FIG. 7C), demonstratingthe important role of NME2 in utilizing cytidine to mediate ECproliferation.

The effect of NME2 can be confirmed with its specific inhibitor ellagicacid (EA) (Malmquist, et al., Proc. West. Pharmacol. Soc., 2001:57-60(1998)).

EA blocked cytidine-restored EC proliferation in a dose-dependentmanner. These results indicate that NME2 protects ECs from CPECchallenge through utilizing cytidine as the substrate for CTP salvagesynthesis pathway. To test if NME2 is involved in there-endothelialization in vivo, NME2 expression was examined in arteriesundergoing vascular repair following injury-induced vascular remodelingImmunohistochemistry revealed that NME2 was expressed mainly in ECs butnot SMCs in arteries.

CPEC treatment induced a strong NME2 expression in ECs of the arterieswith enhanced re-endothelialization, indicating that NME2 may beimportant for the re-endothelialization. To test this, NME2 expressionwas examined in arteries undergoing vascular repair after injury-inducedvascular remodeling. NME2 was expressed mainly in ECs, but not SMCs, inarteries. CPEC treatment induced a strong NME2 expression in ECs of thearteries with enhanced re-endothelialization, indicating that NME2 maybe important for the re-endothelialization (FIG. 7G).

Additionally, both CTPS1 and NME2 were knocked down using shRNAs ininjured rat carotid arteries Immunohistochemistry revealed thatknockdown of CTPS1 and NME2 simultaneously suppressedre-endothelialization as indicated by the loss of CD31 positive cells ascompared to CTPS1 knockdown alone (FIG. 7H). In agreement with theeffect of NME2 knockdown, blockade of NME2 activity by EA administration(10 mg/kg/day) dramatically impaired the re-endothelialization ofwire-injured mouse carotid artery in the presence of CPEC. These datademonstrate that NME2 is important for the proliferation of ECs when thecells are treated with CPEC, which facilitates the re-endothelializationin vivo.

Using both pharmacological and loss of function approaches, Examples 1-6collectively demonstrate that targeting CTPS1 can effectively suppressneointima formation following vascular injury. Importantly, low dosagesof CPEC inhibit SMC, but not EC growth, indicating that ECs are lesssensitive to CPEC treatment. High dosage of CPEC suppresses both SMC andEC proliferation, indicating that a relatively low level of CTPSactivity is required for EC proliferation. The inhibitory effect of highdosage of CPEC can be reversed by addition of cytidine, a substrate forCTP synthesis salvage pathway, indicating that salvage pathway is veryimportant for EC proliferation. Thus, the Examples demonstrate that SMCsand ECs have a distinct preference in utilizing CTP synthesis pathwaysfor their proliferation. SMCs appear to mainly utilize CTPS-mediated denovo synthesis pathway. Although CTPS-mediated pathway is much lessessential for ECs, ECs appear to use both de novo and salvage pathwaysto synthesize CTP.

Examples 1-6 are supportive of a strategy to potentially overcome along-standing medical challenge, i.e., the impairedre-endothelialization in anti-neointima therapy following cardiovascularinterventions. Drug-eluting stents (DES) are a common treatment forcoronary artery diseases. However, drugs currently used in clinic suchas sirolimus-(Cypher) and paclitaxel-(Taxus) have side effects causingdefective re-endothelialization and increasing risk of late thrombosis(Finn, et al., Circulation., 115:2435-2441 (2007)). The results ofExamples 1-6 collectively indicate that blockade of CTPS1 functionaccelerates re-endothelialization in two injury models, which is likelydue to the reduction of proliferating SMCs as well as the induction ofNME-mediated salvage pathways. Proliferative SMCs are known to producevarious paracrine factors such as endostatin and thrombospondin toinhibit EC proliferation (Dhanabal, et al., J. Biol. Chem.,274:11721-11726 (1999); Stouffer, et al., Circulation, 97:907-915(1998); Dawson, et al., J Cell Biol., 138:707-717 (1997)). Reduction ofproliferating SMCs benefits re-endothelialization as long as ECproliferation is not inhibited, which can be achieved via the activationof CTP salvage pathway. Salvage pathway enzymes NME2 appears to beexpressed at a relatively low level in normal EC growth condition.However, when CTPS activity is blocked by CPEC, NME2 is dramaticallyinduced in ECs while neointima proliferating SMCs are significantlyreduced.

Although both NME1 and NME2 are important for CTP synthesis, NME2appears to have a stronger capability in utilizing cytidine. Indeed,knockdown of NME2 significantly diminishes EC proliferation in thepresence of CPEC and cytidine. These results demonstrate that thesalvage pathway is up-regulated in order to compensate for CPEC-blockedCTP synthesis, which sustains the EC proliferation. The combined effectof blocking CTPS1 on inhibiting SMC proliferation while sustaining ECproliferation results in the accelerated re-endothelialization inEC-denuded vessels.

It is interesting that ECs, but not SMCs, utilize salvage pathways tosynthesize CTP, which is likely due to the accessibility of ECs to thesalvage pathway-specific substrate cytidine. Cytidine is circulating inthe blood stream, which consistently interacts with ECs. Therefore, ECsmay adopt a unique mechanism by which cytidine is used to synthesize CTPeven under normal growing or quiescent states. This specialized abilityis obviously enhanced when the de novo pathway is impaired.

Targeting CTPS is likely to result in significantly-improved vascularrepair. In addition to CPEC, several other inhibitors including3-deazauridine (3-DU) (Gao, et al., Nucleosides, Nucleotides NucleicAcids, 19:371-377 (2000)) carbodine (Georges-Courbot, et al., AgentsChemother., 50:1768-1772 (2006)) and 6-diazo-5-oxonorleucine (DON)(Bearne, et al., Biochem. J., 356:223 (2001) also effectively block CTPSactivity, which further make CTPS as a promising target in curingproliferative vascular diseases including those observed incardiovascular interventions.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A method of reducing proliferation of vascular smoothmuscle cells or reducing neointima formation in a subject comprisingimplanting into the subject a medical device comprising an effectiveamount of a CTP synthase 1 (CTPS1) inhibitor to reduce proliferation ofvascular smooth muscle cells (VSMC) in the subject to a greater degreethan the inhibitor reduces endothelial cell proliferation in thesubject, wherein the CTPS1 inhibitor is a nucleoside analog.
 2. Themethod of claim 1, wherein the amount of CTPS1 inhibitor is notsufficient to reduce proliferation of endothelial cells in the subject.3. The method of claim 1, wherein the degree to which VSMC proliferationis reduced relative to EC proliferation is effective to (i) reduceneointima formation at the site of implantation of the device in thesubject, (ii) permit or promote re-endothelialization at the site ofimplantation of the device in the subject, or (iii) a combinationthereof.
 4. The method of claim 1, wherein nucleoside analog is selectedfrom the group consisting of cyclopentenyl cytosine (CPEC),cyclopentenyl cytosine 5′-triphosphate (CPEC-TP), 3-deazauridine (3-DU),(+) carbodine, (−) carbodine, and pharmacologically active saltsthereof.
 5. The method of claim 3, wherein nucleoside analog is selectedfrom the group consisting of cyclopentenyl cytosine (CPEC),cyclopentenyl cytosine 5′-triphosphate (CPEC-TP), 3-deazauridine (3-DU),(+) carbodine, (−) carbodine, and pharmacologically active saltsthereof.
 6. The method of claim 1, wherein the CTPS1 inhibitor isincorporated into or encapsulated in a delivery vehicle for deliveringthe CTPS1 inhibitor to vascular smooth muscle cells.
 7. The method ofclaim 6, wherein the delivery vehicle is selected from the groupconsisting of nanoparticles, microparticles, micelles, syntheticlipoprotein particles, liposomes, and carbon nanotubes.
 8. The method ofclaim 1, wherein a targeting signal for enhancing delivery of the CTPS1inhibitor to vascular smooth muscle cells is (i) operably linked to theCTPS1 inhibitor, or (ii) operably linked to the delivery vehicle fordelivering the CTPS1 inhibitor to vascular smooth muscle cells.
 9. Themethod of claim 8, wherein the targeting signal binds to Tissue Factor,α_(v)β₃ integrin, or a marker of a clot or thrombosis selected from thegroup consisting of fibrin, gpIIb/IIIa, tissue factor/VIIA complex,activated clotting factor Xa, activated clotting factor IXa, and thefibrin condensation product d-dimer.
 10. The method of claim 1, whereinthe CTPS1 inhibitor is coated onto or incorporated into the device. 11.The method of claim 1, wherein the device is selected from the groupconsisting of implants, stents, and valves.
 12. The method of claim 11wherein the device is a drug eluting stent that elutes the CTPS1inhibitor or a delivery vehicle comprising the CTPS1 inhibitor.
 13. Themethod of claim 1, wherein the site of implantation or a site adjacentthereto comprises an injury in need of re-endothelialization, andwherein re-endothelialization occurs faster in the presence of thedevice than a control device without the CTPS1 inhibitor.
 14. The methodof claim 1, wherein the subject has a vascular proliferation disorder orvascular trauma, or has undergone or is undergoing angioplasty, vascularsurgery, or transplantation arteriopathy.
 15. The method of claim 1further comprising administering to the subject cytidine or a cytidineanalog.
 16. The method of claim 1, wherein the amount of CTPS1 inhibitorreduces proliferation of VSMC to a greater degree than the inhibitorreduces EC proliferation in an in vitro cell proliferation assay. 17.The method of claim 1, wherein the amount of CTPS1 inhibitor reducesproliferation of VSMC to a greater degree than the inhibitor reduces ECin a rodent carotid artery wire injury model.
 18. The method of claim 1,wherein the VSMC are media layer VSMC.
 19. A method of reducingproliferation of vascular smooth muscle cells or reducing neointimaformation in a subject comprising implanting into the subject a medicaldevice comprising an effective amount of cyclopentenyl cytosine (CPEC)to reduce proliferation of vascular smooth muscle cells (VSMC) in thesubject to a greater degree than the CPEC reduces endothelial cellproliferation in the subject.
 20. The method of claim 19, wherein theVSMC are media layer VSMC, and the degree to which the media layer VSMCproliferation is reduced relative to the EC is effective to (i) reduceneointima formation at the site of implantation of the device in thesubject, (ii) permit or promote re-endothelialization at the site ofimplantation of the device in the subject, or (iii) a combinationthereof.